Culture method of bile duct cancer organoid and phenotype identification method of immunofluorescence of bile duct cancer organoid

Abstract

The invention relates to a culture method of bile duct cancer organoid. The culture method comprises the following steps: S1, cleaning bile duct cancer tissues in a biosafety cabinet, and chopping the bile duct cancer tissues; s2, transferring the chopped tissue into a centrifugal tube filled with tissue digestive juice for digestion, and collecting an upper suspension; s3, centrifuging the collected upper suspension, and collecting bile duct cancer tissue cell precipitate; s4, inoculating and embedding bile duct cancer tissue cells into matrigel; s5, after the matrigel is solidified, culturing the tissue cells by using a corresponding organoid culture medium, and obtaining primary organoid; the culture medium is replaced every three days. Due to the adoption of the technical scheme, the culture method of the bile duct cancer organoid can be used for efficiently and stably culturing the bile duct cancer organoid.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically to a simple and stable culture medium for long-term culture of cholangiocarcinoma organoids, as well as a culture method for cholangiocarcinoma organoids and an immunofluorescence phenotypic identification method. Background Technology

[0002] Cholangiocarcinoma (CCA) is a highly malignant tumor with a poor prognosis. 50% of patients with biliary malignancies are diagnosed at an advanced stage, with a survival of less than one year. Only about 10% of patients are eligible for surgery at the time of diagnosis, and the recurrence rate within one year after surgery is as high as 67%, with a 5-year survival rate of 5%–15%. Individual differences exist in the expression of genetic information, gene information, and molecular levels in cholangiocarcinoma among different patients, which also affects patient prognosis. The search for new treatment methods and new cholangiocarcinoma models is urgently needed.

[0003] Organoids are organ-specific 3D cell models that can spontaneously assemble and grow in vitro. Derived from autologous tissues, organoids can highly replicate the specific genetic information of tissues and organs, and partially reproduce their functions. Organoids have significant applications in disease model construction, drug screening, and personalized medicine.

[0004] Currently, existing culture media for cholangiocarcinoma organoids are relatively complex in composition and their culture systems are not mature enough. Therefore, there is an urgent need for a highly efficient, stable, and simple culture medium for cholangiocarcinoma organoid models that can be used for long-term stable culture of cholangiocarcinoma organoids for disease research and drug development. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an efficient and stable method for culturing bile duct carcinoma organoids.

[0006] This invention is achieved through the following technical solution: A method for culturing bile duct carcinoma organoids, comprising the following steps: S1. Clean the bile duct cancer tissue in a biosafety cabinet and then chop the bile duct cancer tissue into small pieces; S2. Transfer the chopped tissue to a centrifuge tube containing tissue digestion solution for digestion, and collect the supernatant suspension. S3. After centrifuging the collected upper suspension, collect the cholangiocarcinoma tissue cell precipitate. S4. Inoculate cholangiocarcinoma tissue cells into matrix gel. S5. After the matrix gel solidifies, culture the tissue cells using the corresponding organoid culture medium to obtain primary organoids. Change the culture medium every 3 days.

[0007] Furthermore, after step S5, the following steps are also included: S6. When the density of primary organoids reaches 80%-90% of the matrix gel space, add ice-cooled DMEM washing medium, and use a cold pipette tip to dissociate the matrix gel, or place the 24-well plate in a -20°C freezer for 4 minutes to aid in matrix gel dissociation. Then collect the mixture in the wells into a 15 ml centrifuge tube, add 2-3 times the volume of ice-cooled DMEM washing medium to the centrifuge tube to dilute the matrix gel and organoid mixture, and centrifuge the mixture at 4°C and 300×g for 5 minutes. S7. After centrifugation, remove the supernatant, add 1 ml TrypLE Express enzyme to the organoid precipitate, and incubate the mixture in a rotary thermostat at 37°C and 30 rpm for 10 min. Add 10 ml of ice-cooled Advanced DMEM / F-12 washing medium to stop digestion, and centrifuge the mixture at 4°C and 300×g for 5 min. S8. After centrifugation, observe whether the organoids have completely precipitated and whether there is any unremoved matrix gel-primary organoid mixture in the intermediate layer. If only obvious cells or cell clusters are observed to precipitate, remove the supernatant, add cold, fresh Cultrex Reduced Growth Factor Basement Membrane Extract (type 2) to resuspend the precipitate, and re-inoculate at a ratio of 1:2 to 1:4. Use a pipette to add the precipitate to each well of a 24-well plate. Invert the 24-well plate and incubate it in a sterile cell culture incubator at 37°C with 5% CO2 for about 30 minutes until it solidifies into a dome shape. After the Basement Membrane Extract has solidified, add cholangiocarcinoma organoid culture medium for culturing; or add organoid cryopreservation solution to the precipitate and freeze it in a liquid nitrogen tank for long-term storage.

[0008] Furthermore, the tissue cleaning process in step S1 includes the following steps: S11. Place the obtained cholangiocarcinoma tissue into a 10cm cell culture dish; S12. Add 10 ml of DMEM washing medium containing 1% FBS, 1% Penicillin-Streptomycin-Neomycin, and 100 µg / ml Primocin to the culture dish to wash the tissue.

[0009] Furthermore, the tissue digestion solution in step S2 comprises the following components: based on Advanced DMEM / F-12 medium, with the following final concentrations added: 10 mM HEPEs, 1x GlutaMAX Supplement, 100 µg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin, 2.5% FBS, 5 mg / ml Collagenase II, 1 mg / ml Dispase II, 10.5 µM Y-27632, and 10 µg / ml DNAse I.

[0010] Furthermore, the cleaning and shredding of bile duct cancer tissue within the biosafety cabinet are performed with the assistance of a biosafety cabinet auxiliary device used to limit the speed of the operator's forearm movement within the biosafety cabinet.

[0011] Furthermore, the biosafety cabinet uses an auxiliary device including a fixing plate, a first speed limiting part, and a second speed limiting part. The fixing plate is used to fix the biosafety cabinet to the front side wall. The first limiting part and the second speed limiting part are both located inside the biosafety cabinet and are disposed on the fixing plate. The first speed limiting part is used to fix and connect the front end of the operator's forearm and to limit the speed of the operator's forearm moving horizontally and downward. The second speed limiting part is used to hold the operator's forearm downward and to limit the speed of the operator's forearm moving upward.

[0012] Furthermore, the first speed limiting part includes a horizontal plate, a tube, a first piston, a pull rope, a fixing part, and a driving part. The front end of the horizontal plate is fixed on the horizontal plate, and the rear end extends horizontally backward. A through hole is formed at the rear end of the horizontal plate. The first end and the second end of the tube are both closed. A perforation is formed on the end wall of the first end of the tube. The first piston slides in the tube and divides the inner cavity of the tube into a first cavity and a second cavity. Both the first cavity and the second cavity are filled with liquid. A first connecting hole is formed on the first piston to connect the first cavity and the second cavity. The lower end of the pull rope passes downward through the through hole, and the upper end of the pull rope passes through the perforation into the first cavity and is fixedly connected to the first piston. The pull rope slides in the perforation. The fixing part is fixedly connected to the lower end of the pull rope. The fixing part is used to fix the front end of the operator's forearm. The driving part is used to drive the first piston to move away from the first end wall of the tube during the movement of the patient's forearm towards the axis of the through hole.

[0013] Furthermore, the tube body includes a horizontal section, a curved section, and a vertical section. The horizontal section is parallel to the front sidewall of the biosafety cabinet and is fixedly connected to the fixing plate. The first end of the horizontal section is the first end of the tube body. The upper end of the curved section is fixedly connected to the second end of the horizontal section. The upper end of the vertical section is fixedly connected to the lower end of the curved section. The lower end of the vertical section is the second end of the tube body. The first piston is slidably fitted within the horizontal section. The first speed limiting part also includes a first steering wheel and a second steering wheel. Both the first steering wheel and the second steering wheel are rotatably connected to the fixed plate. The rotation center line of the first steering wheel is set along the left and right direction, and the rotation center line of the second steering wheel is set along the vertical direction. The drive unit includes a connecting rope and a counterweight ball. One end of the connecting rope is fixedly connected to the first piston, and the other end passes through the curved section and is located at the lower end of the vertical section. The counterweight ball is located inside the vertical section and is fixedly connected to the other end of the connecting rope. The upper end of the pull rope passes sequentially around the rear side of the first deflector wheel and the front side of the second deflector wheel before passing through the hole.

[0014] Furthermore, the lower end of the through hole wall and the lower side surface of the horizontal plate form a rounded transition.

[0015] Furthermore, the second speed limiting part includes a vertical tube, a second piston, a lifting rod, and a pressure rod. The vertical tube is fixedly connected to the fixing plate, and both the upper and lower ends of the vertical tube are closed. A circular hole is formed on the lower end wall of the vertical tube. The second piston is slidably fitted inside the vertical tube, dividing the inner cavity of the vertical tube into an upper cavity and a lower cavity. Both the upper cavity and the lower cavity are filled with liquid. A second connecting hole is formed on the second piston to connect the upper cavity and the lower cavity. The upper end of the lifting rod passes through the circular hole into the lower cavity and connects to the second piston. The lifting rod is slidably fitted inside the circular hole. The pressure rod is fixed to the lower end of the lifting rod and located on the rear side of the fixing part. The pressure rod is arranged in the left-right direction.

[0016] The beneficial effects of this invention are as follows: The method for culturing cholangiocarcinoma organoids described in this invention can efficiently and stably culture cholangiocarcinoma organoids.

[0017] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0018] Figure 1 This is a flowchart of the method for culturing cholangiocarcinoma organoids according to the present invention; Figure 2 Observation of tissue morphology and structure of primary cultured cholangiocarcinoma organoids under a regular optical microscope; Figure 3 To observe tissue morphology and structure in different wells under a regular optical microscope for subsequent long-term passage culture (ninth generation); Figure 4 To facilitate long-term passage culture (ninth generation), we will observe the tissue morphology and structure and organoid growth process daily under a regular optical microscope.

[0019] Figure 5 Multichannel imaging of bile duct carcinoma organoids in an immunofluorescence assay. Figure 6 This is a schematic diagram of the auxiliary device used in the biosafety cabinet in the cholangiocarcinoma organoid culture method of the present invention; Figure 7 for Figure 6 Enlarged view of 'a' in the middle; Figure 8 This is a schematic diagram of the combined structure of the tube body, pull rope, first piston, connecting rope and counterweight ball in the auxiliary device for using a biosafety cabinet in the cholangiocarcinoma organoid culture method of the present invention. Figure 9 This is a top view of the combined structure of the tube body, pull rope, first piston, connecting rope and counterweight ball in the auxiliary device for using a biosafety cabinet in the cholangiocarcinoma organoid culture method of the present invention. Figure 10 For Figure 9 AA section view; Figure 11 for Figure 10 Enlarged view of b in the middle; Figure 12 for Figure 10 Enlarged view of C in the middle; Figure 13 This is a schematic diagram of the second speed limiting part in the auxiliary device for using a biosafety cabinet in the method for culturing cholangiocarcinoma organoids of the present invention; Figure 14 This is a top view of the second speed limiting part in the auxiliary device for using a biosafety cabinet in the method for culturing cholangiocarcinoma organoids of the present invention; Figure 15 for Figure 14 BB section view; Figure 16 for Figure 15 Enlarged view of d in the middle; Figure 17 for Figure 15 Enlarged view of 'e' in the middle; Figure 18A schematic diagram of the structure of a 24-well plate I with a concave hydrophilic cavity; Figure 19 A top view of a 24-well plate I with a concave hydrophilic cavity; Figure 20 for Figure 19 AA section view; Figure 21 A schematic diagram of the structure of a 24-well plate II with a concave hydrophilic cavity; Figure 22 A top view of a 24-well plate II with a concave hydrophilic cavity; Figure 23 for Figure 19 BB cross-sectional view.

[0020] In the diagram: Fixed plate-1, Horizontal plate-2, First piston-3, Pull rope-4, Fixed block-5, Strap-6, Hook and loop fastener face-7, Hook and loop fastener rough face-8, First sealing ring-9, Horizontal section-10, Curved section-11, Vertical section-12, First directional wheel-13, Second directional wheel-14, Connecting rope-15, Counterweight ball-16, First bracket-17, Second bracket-18, First shaft-19, Second shaft-20, Vertical tube-21, Second piston-22, Lifting rod-23, Pressure rod-24, Connecting rod-25, Second sealing ring-26. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0022] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0023] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0024] In the above description of the present invention, it should be noted that the terms "one side," "the other side," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship in which the product of the invention is conventionally placed during use. These terms are used only for the convenience of describing the present invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0025] Furthermore, terms such as "identical" do not imply that components must be absolutely identical; minor differences are permissible. The term "perpendicular" simply means that the positional relationship between components is more perpendicular than "parallel," not that the structure must be perfectly perpendicular; a slight tilt is acceptable.

[0026] Please see Figure 1 This invention provides a technical solution: a method for culturing bile duct carcinoma organoids, comprising the following steps: S1. Clean the bile duct cancer tissue in a biosafety cabinet and then chop the bile duct cancer tissue into small pieces; The tissue in step S1 is tissue that has been surgically removed.

[0027] In this embodiment, the tissue cleaning in step S1 includes the following steps: S11. Place the obtained cholangiocarcinoma tissue into a 10cm cell culture dish; S12. Add 10 ml of DMEM washing medium containing 1% FBS, 1% Penicillin-Streptomycin-Neomycin, and 100 µg / ml Primocin to the culture dish to wash the tissue. This washing process removes impurities such as fat.

[0028] In a preferred embodiment, the chopping mentioned in step S1 refers to placing the tissue block in a cell culture dish and chopping it into minced meat-like fragments using a disposable scalpel.

[0029] S2. Transfer the chopped tissue to a centrifuge tube containing tissue digestion solution for digestion, and collect the supernatant suspension. The tissue digestion solution in step S2 comprises the following components: based on Advanced DMEM / F-12 medium, with the following final concentrations added: 10 mM HEPEs, 1x GlutaMAX Supplement, 100 µg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin, 2.5% FBS, 5 mg / ml Collagenase II, 1 mg / ml Dispase II, 10.5 µM Y-27632, and 10 µg / ml DNAse I.

[0030] The digestion described in step S2 involves placing the tissue fragments in a centrifuge tube containing 3 ml of digestion solution and performing the digestion process in a rotary thermostat mixer at a temperature of 37°C and a rotation speed of 30 rpm.

[0031] In a preferred embodiment, the digestion of the chopped tissue in step S2 is carried out by repeated digestion. For the first digestion: place the tissue fragments in a centrifuge tube containing 3 ml of digestion solution, and digest them in a rotary thermostat at 37°C and 30 rpm for no more than 15 min. Then, let them stand under gravity for 1 min and collect the supernatant. Finally, add 3-4 times the volume of DMEM washing medium to terminate the digestion.

[0032] For each digestion after the first digestion: add 1-2 ml of tissue digestion solution to the tissue precipitate left after the previous digestion and place it in a rotary thermostat mixer. Maintain the temperature at 37°C and the rotation speed at 30 rpm for 5-10 min to obtain a turbid solution. After standing for 1 min under gravity, collect the supernatant and then add 3-4 times the volume of DMEM washing medium to terminate the digestion.

[0033] The chopped tissue should be digested repeatedly until most of it is digested, leaving only a small amount of tissue fragments. In each subsequent digestion after the first digestion, the amount of digestive fluid can be reduced by half. Multiple digestions can prevent overdigestion. Overdigestion here refers to cells being digested by the digestive fluid for too long, resulting in poor cell growth or even preventing cell growth.

[0034] S3. After centrifuging the collected upper suspension, collect the cholangiocarcinoma tissue cell precipitate. In a preferred embodiment, the process described in step S3 includes the following steps: S31. Centrifuge the supernatant collected in step S2 at 4°C and 300g for 5 min, discard the supernatant, and keep the cell pellet.

[0035] S32. Then, resuspend the cell and tissue pellet obtained in step S31 in 10 ml of the above washing medium, gently blow and aspirate it several times with a wide-mouth pipette tip, centrifuge at 300g for 5 min at 4°C, remove the supernatant, and obtain the cell and tissue pellet.

[0036] S33. Repeat step S32 once from the cell and tissue precipitate obtained in step S32 to obtain cell and tissue precipitate.

[0037] S34. Resuspend the cell and tissue pellet obtained in step S33 in 10 ml of Advanced DMEM / F-12 culture medium, gently pipette it a few times with a wide-mouth pipette tip, centrifuge at 300g for 5 min at 4℃, and remove the supernatant as thoroughly as possible to obtain the cell and tissue pellet.

[0038] S4. Inoculate cholangiocarcinoma tissue cells into matrix gel. As a preferred embodiment, the matrix adhesive embedding in step S4 can be applied in the center of the well plate at a rate of 30 µl per drop, with 1 drop per well.

[0039] As a preferred embodiment, the matrix gel embedding in step S4 involves resuspending the centrifuged cell / tissue pellet with Cultrex Reduced Growth Factor Basement Membrane Extract, type 2, adding it to a 24-well plate, and incubating the plate upside down in a sterile cell culture incubator at 37°C and 5% CO2, so that the matrix gel embedding the tissue cells solidifies into a dome shape.

[0040] S5. After the matrix gel solidifies, culture the tissue cells using the corresponding organoid culture medium to obtain primary organoids. Change the culture medium every 3 days. The tissue in question is cholangiocarcinoma tissue.

[0041] The organoid culture medium in step S5 comprises the following components: based on Advanced DMEM / F-12 medium, the following components are added to a final concentration: 10% (V / V) R-spondin 1-Conditioned Medium, 10 mM HEPEs, 1x GlutaMAX Supplement, 100 µg / ml Primocin, 1% (V / V) Penicillin-Streptomycin-Neomycin, 2.5% FBS, 2% (V / V) B-27 supplement (50x), 1% (V / V) N2 supplement (100x), 10 mM Nicotinamide, 1.25 mM N-acetylcysteine, 50 ng / ml mEGF, 10 nM hGastrin I, 5 µMA83-01, 10 µM forskolin, and 10.5 µM Y-27632. Y-27632 was added during the initial culture, passage, and revival processes.

[0042] As a preferred embodiment, the culture described in step S5 involves placing the 24-well plate after dispensing the adhesive in a sterile cell culture incubator at 37°C and 5% CO2.

[0043] In a preferred embodiment, the curing time of the matrix adhesive in step S5 depends on the size of the droplets when dispensing the adhesive, and is usually greater than 20 minutes.

[0044] In a preferred embodiment, the following steps are included after step S5: S6. When the primary organoid density reaches 80%-90% of the matrix gel space, add ice-cooled DMEM washing medium and use a cold pipette tip to dissociate the matrix gel, or place the 24-well plate in a -20°C freezer for 4 minutes to aid in matrix gel dissociation. Then collect the mixture from the wells into a 15 ml centrifuge tube, add 2-3 times the volume of ice-cooled DMEM washing medium to the centrifuge tube to dilute the matrix gel and organoid mixture, and centrifuge the mixture at 4°C and 300×g for 5 minutes.

[0045] S7. After centrifugation, remove the supernatant and add 1 ml of TrypLE Express enzyme to the organoid precipitate. Incubate the mixture in a rotary oscillator at 37°C and 30 rpm for 10 min. Add 10 ml of ice-cooled Advanced DMEM / F-12 wash medium to terminate digestion. Centrifuge the mixture at 4°C and 300×g for 5 min.

[0046] S8. After centrifugation, observe whether the organoids have completely precipitated and whether there is any unremoved matrix gel-primary organoid mixture in the intermediate layer. If only obvious cell or cell cluster precipitation is observed, remove the supernatant, add cold, fresh Cultrex Reduced Growth Factor Basement Membrane Extract (type 2) to resuspend the precipitate, and re-seed it at a ratio of 1:2 to 1:4. Carefully add the precipitate to each well of a 24-well plate using a pipette. Invert the 24-well plate and incubate it in a sterile cell culture incubator at 37°C with 5% CO2 for about 30 minutes until it solidifies into a dome shape. After the Basement Membrane Extract matrix gel has solidified, add it to the cholangiocarcinoma organoid culture medium for culture; or add organoid cryopreservation solution to the precipitate and freeze it in a liquid nitrogen tank for long-term storage.

[0047] The solution preparation involved in this embodiment is as follows: The tissue preservation solution was prepared from the following components: DMEM, 1% Penicillin-Streptomycin-Neomycin, and 100 µg / ml Primocin.

[0048] The tissue washing solution was prepared from the following components: PBS, 1% Penicillin-Streptomycin-Neomycin, and 100 µg / ml Primocin.

[0049] The tissue cryopreservation solution is prepared from the following components: 10% DMSO and 90% FBS.

[0050] The tissue digestion solution was prepared from the following components: 10 mM HEPEs, 1x GlutaMAX Supplement, 100 µg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin, 2.5% FBS, 5 mg / ml Collagenase II, 1 mg / ml Dispase II, 10.5 µM Y-27632, and 10 µg / ml DNAse I in Advanced DMEM / F-12 medium.

[0051] DMEM wash medium is prepared from the following components: 1% FBS, 1% Penicillin-Streptomycin-Neomycin, and 100 µg / ml Primocin in DMEM medium.

[0052] Advanced DMEM / F-12 wash medium is prepared from the following components: 10 mM HEPEs, 1xGlutaMAX Supplement, 100 µg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin, and 2.5% FBS.

[0053] The culture medium for cholangiocarcinoma organoids was prepared from the following components: Advanced DMEM / F-12 medium supplemented with 10% (V / V) R-spondin1 R-spondin1-Conditioned Medium, 10 mM HEPEs, 1x GlutaMAX Supplement, 100 µg / ml Primocin, 1% (V / V) Penicillin-Streptomycin-Neomycin, 2.5% FBS, 2% (V / V) B-27 supplement (50x), 1% (V / V) N2 supplement (100x), 10 mM Nicotinamide, 1.25 mM N-acetylcysteine, 50 ng / ml hEGF, 10 nM hGastrin I, 5 µM A83-01, 10 µM forskolin, and 10.5 µM Y-27632.

[0054] The organoid cryopreservation solution is prepared from the following components: 90% (V / V) FBS and 10% (V / V) DMSO.

[0055] Cultivation Experiment 1 S1. Obtain bile duct cancer tumor tissue from the affected area from clinical personnel, place it in tissue preservation solution, and transfer it from the operating room to the laboratory biosafety cabinet for processing. Use disposable forceps to remove the tissue from the preservation solution and place it in sterile tissue washing solution, washing three times. Use a disposable sterile scalpel to divide the tissue into multiple 2-3 mm sections. 3 Small pieces. Randomly select 3-5 tissue pieces and place them in cryovials, add tissue cryopreservation solution, place them in a cryopreservation box and store at -80°C for 48 hours, then transfer them to a liquid nitrogen tank for preservation. Randomly select one piece of tissue from the cut tumor tissue and mince it with a disposable sterile scalpel until it is in a paste-like state.

[0056] S2. Collect bile duct carcinoma tissue fragments into 5 ml centrifuge tubes, add 3 ml of tissue digestion solution, and digest in a rotary thermostat at 37°C and 30 rpm for 15 min. Then place on moist ice and let stand for 1 min. Collect the supernatant suspension under gravity into a 15 ml centrifuge tube, add 3-4 times the volume of the digestion solution in ice-cooled washing medium, and terminate digestion on ice. If a large amount of tissue fragments remain, repeat digestion multiple times until most of the tissue is digested, leaving only a small amount of tissue fragments. Collect the supernatant suspension and cell / tissue precipitate.

[0057] S3. After centrifuging the collected upper suspension, collect the cholangiocarcinoma tissue cell precipitate.

[0058] S31. Centrifuge the upper suspension collected in step S2 at 4°C and 300 g for 5 min to remove the supernatant and obtain cell and tissue precipitates.

[0059] S32. Then, resuspend the cell and tissue pellet obtained in step S31 in 10 ml of the above washing medium, gently blow and aspirate it several times with a wide-mouth pipette tip, centrifuge at 300×g for 5 min at 4°C, remove the supernatant, and obtain the cell and tissue pellet.

[0060] S33. Repeat step S32 once from the cell and tissue precipitate obtained in step S32 to obtain cell and tissue precipitate.

[0061] S34. Resuspend the cell and tissue pellet obtained in step S33 in 10 ml of Advanced DMEM / F-12 culture medium, gently pipette it a few times with a wide-mouth pipette tip, centrifuge at 300×g for 5 min at 4℃, and remove the supernatant as thoroughly as possible to obtain the cell and tissue pellet.

[0062] S4. Inoculate cholangiocarcinoma tissue cells into matrix gel. Specifically, the cell / tissue pellet obtained in step S34 was directly resuspended in Cultrex Reduced Growth Factor Basement Membrane Extract, type 2 (Matrix Gel). The pellet was carefully added to a 24-well plate using a pipette. Matrix Gel was dropped into the center of each well at a rate of 30 µl, one drop per well. The 24-well plate was then inverted and placed in a sterile cell culture incubator at 37°C with 5% CO2 aeration for approximately 30 minutes, allowing the matrix gel containing the embedded tissue cells to solidify in a dome shape.

[0063] For standard 24-well plates, the plates usually need to be inverted to allow the matrix gel to form a 3D droplet space under the influence of gravity and droplet tension. As a preferred method, cell / tissue pellets resuspended in the matrix gel can be added to a 24-well plate (Type I) with a concave hydrophilic cavity. The plate can then be placed upright in a sterile cell culture incubator at 37°C with 5% CO2 aeration for approximately 30 minutes. Due to the volume limitation of the concave hydrophilic cavity, the matrix gel will solidify into a 3D cylindrical space (please refer to...). Figure 18-20 Compared to ordinary 24-well plates, when placed upright, the matrix gel containing cells / tissues will spread evenly at the bottom of the 24-well plate under gravity. The 24-well plate I with concave hydrophilic cavities will avoid the above problems.

[0064] In a 24-well plate I with a concave hydrophilic cavity, the small top area of ​​the matrix gel, after solidification within the grooves, results in a small contact surface with the culture medium, hindering nutrient exchange. As a preferred method, cell / tissue pellets resuspended in the matrix gel can be added to a 24-well plate II with a concave hydrophilic cavity and incubated in a sterile cell culture incubator at 37°C and 5% CO2 for approximately 30 minutes. Due to the volume limitation of the concave hydrophilic cavity, the matrix gel will solidify into a 3D space resembling a frustum (see reference). Figure 21-23 This creates a solid cross-section that is larger at the top and smaller at the bottom, which will further increase the contact area between the solid matrix gel and the culture medium, thus facilitating the exchange of substances.

[0065] S5. After the matrix gel solidifies, culture the tissue cells using the corresponding organoid culture medium to obtain primary organoids. Change the culture medium every 3 days. Specifically, the curing time of the matrix gel depends on the size of the droplets during dispensing, and usually takes more than 20 minutes. The cells are cultured for 7-10 days in a sterile cell culture incubator at 37℃ with 5% CO2 aeration. During the culture process, the cholangiocarcinoma organoid culture medium is replaced every 3 days, with the replacement medium containing cholangiocarcinoma organoids without Y-27632 added. The growth status of the organoids is observed daily, and photographs are taken and recorded under a high-content microscope to obtain primary cholangiocarcinoma organoids.

[0066] Please refer to the following: Figure 2 This is an example of observing the tissue morphology and structure of primary cultured cholangiocarcinoma organoids under a regular optical microscope. Specifically, it is an example of the primary (P0) culture growth process of cholangiocarcinoma organoids. On day 2 of growth, obvious organoids (B) were observed. With continued growth, the diameter of the organoids increased significantly, with some larger organoids reaching approximately 1 mm in diameter. Typically, they reach passage level after 7-10 days of growth.

[0067] S6. When the primary organoids have been cultured for 10 days and the density of primary organoids in the 24-well plate reaches 80%-90% of the matrix gel space, remove the original culture medium and add ice-cooled DMEM wash medium. Use a cold pipette tip to dissociate the matrix gel or place the 24-well plate in a -20°C freezer for 4 minutes to aid in matrix gel dissociation. Then collect the mixture in the wells into a 15 ml centrifuge tube. Add 2-3 times the volume of ice-cooled DMEM wash medium to the centrifuge tube to dilute the matrix gel and organoid mixture. Centrifuge the mixture at 4°C and 300 × g for 5 min.

[0068] S7. After centrifugation, remove the supernatant and add 1 ml of TrypLE Express enzyme to the organoid precipitate. Incubate the mixture in a rotary oscillator at 37°C and 30 rpm for 10 min. Add 10 ml of ice-cooled Advanced DMEM / F-12 wash medium to terminate digestion. Centrifuge the mixture at 4°C and 300×g for 5 min.

[0069] S8. After centrifugation, observe whether the organoids have completely precipitated and whether there is any unremoved matrix gel-primary organoid mixture in the intermediate layer. If only obvious cell or cell cluster precipitation is observed, remove the supernatant, add cold, fresh Cultrex Reduced Growth Factor Basement Membrane Extract (type 2) to resuspend the precipitate, and re-seed it at a ratio of 1:2 to 1:4. Carefully add the precipitate to each well of a 24-well plate using a pipette. Invert the 24-well plate and incubate it in a sterile cell culture incubator at 37°C with 5% CO2 for about 30 minutes until it solidifies into a dome shape. After the Basement Membrane Extract matrix gel has solidified, add it to the cholangiocarcinoma organoid culture medium for culture; or add organoid cryopreservation solution to the precipitate and freeze it in a liquid nitrogen tank for long-term storage.

[0070] like Figure 3 The image shows the tissue morphology and structure observed in different wells under a regular optical microscope after long-term passage (ninth generation) culture of a cholangiocarcinoma organoid. This represents the organoid growth in different wells after long-term passage (ninth generation) culture of a single cholangiocarcinoma organoid.

[0071] like Figure 4 The image shows the daily observation of tissue morphology and organoid growth under a regular optical microscope after long-term passage (ninth generation). This is specifically a case of cholangiocarcinoma undergoing long-term passage (ninth generation) culture and continuous observation for 6 days (passivable) to observe organoid growth.

[0072] Phenotypic identification methods using organoid immunofluorescence: Primary organoids were seeded into Matrigel and fixed 2-3 days after seeding.

[0073] Aspirate the culture medium from the culture chamber and rinse once quickly with 200 μL of 1×PBS at room temperature. Fix with 10% formalin (100 μL) at room temperature for 15 minutes. Wash three times with PBS (200 μL), 5 minutes each time.

[0074] Add 200 μL of 0.5% Triton X-100 / PBS permeabilizer and incubate at room temperature for 10 minutes to perform permeabilization. Wash three times with 200 μL PBST (PBS with Tween-20, 0.1%), 3 minutes each time.

[0075] Add rapid sealing solution (200 uL) and seal for 60 minutes.

[0076] Remove the blocking solution, add 100 μl of primary antibody diluted with antibody dilution buffer (see Appendix 1), and incubate at room temperature for 12 h. Remove the primary antibody solution. Wash three times with 1×PBST for 3 minutes each time.

[0077] Add 50 μL of secondary antibody diluted with antibody dilution buffer (see Appendix 1) and incubate at room temperature for 1 h.

[0078] Add 100 μL of anti-fluorescence quenching mounting solution (containing DAPI) (see Appendix 1) and incubate at room temperature for 10 minutes.

[0079] Take photos using a confocal microscope, following the channels. Please refer to the images obtained. Figure 5 .

[0080] Appendix 1 Please see Figure 6-17 In this embodiment, the cleaning and shredding of bile duct cancer tissue in the biosafety cabinet are carried out with the assistance of a biosafety cabinet auxiliary device, which is used to limit the movement speed of the operator's forearm inside the biosafety cabinet.

[0081] When cleaning and shredding cholangiocarcinoma tissue in a biosafety cabinet, auxiliary devices can be used to limit the speed of the operator's forearm movement within the cabinet. This reduces the likelihood of the operator's forearm moving too quickly and minimizes the impact of the operator's forearm on the stability of airflow within the biosafety cabinet.

[0082] In this embodiment, the auxiliary device used in the biosafety cabinet includes a fixing plate 1, a first speed limiting part, and a second speed limiting part. The fixing plate 1 is used to fix the biosafety cabinet to the front side wall. Here, the front side wall of the biosafety cabinet refers to the side wall of the biosafety cabinet opposite to the operating port. In the following description, the front side wall of the biosafety cabinet will have this meaning. The first limiting part and the second speed limiting part are both located inside the biosafety cabinet and are provided on the fixing plate 1. The first speed limiting part is used to fix and connect the front end of the operator's forearm and to limit the speed of the operator's forearm moving horizontally and downward. Here, the front end of the forearm refers to the end of the operator's forearm near the wrist. In the following description, the front end of the forearm will have this meaning. The second speed limiting part is used to hold the operator's forearm downward and to limit the speed of the operator's forearm moving upward.

[0083] In this solution, the fixing plate 1 can be fixed to the front wall of the biosafety cabinet by means of strong adhesive.

[0084] The first speed limiter restricts the horizontal and downward movement speed of the operator's forearm, while the second speed limiter restricts the upward movement speed. Through the combined action of these two speed limiters, the movement speed of the operator's forearm in all directions can be limited. This auxiliary device in the biosafety cabinet effectively limits the movement speed of the operator's forearm within the cabinet.

[0085] In this embodiment, the first speed limiting part includes a horizontal plate 2, a tube body, a first piston 3, a pull rope 4, a fixing part, and a driving part. The front end of the horizontal plate 2 is fixed to the horizontal plate 2, and the rear end extends horizontally backward. A through hole is formed at the rear end of the horizontal plate 2. The first end and the second end of the tube body are both closed. A perforation is formed on the end wall of the first end of the tube body. The first piston 3 is slidably fitted into the tube body and divides the inner cavity of the tube body into a first cavity and a second cavity. Both the first cavity and the second cavity are filled with liquid. A connection is formed on the first piston 3 to the... The first cavity and the second cavity have a first connecting hole. The lower end of the pull rope 4 passes downward through the through hole. The upper end of the pull rope 4 passes through the through hole into the first cavity and is fixedly connected to the first piston 3. The pull rope 4 is slidably fitted in the through hole. The pull rope 4 can be a solid nylon thread with a smooth surface. The fixing part is fixedly connected to the lower end of the pull rope 4. The fixing part is used to fix it to the front end of the operator's forearm. The driving part is used to drive the first piston 3 to move away from the first end wall of the tube body during the movement of the patient's forearm end close to the axis of the through hole.

[0086] In the initial state, there is a certain distance between the first piston 3 and the first end wall of the tube body, and the lower end of the pull rope 4 and the fixing part are both at a certain height from the operating surface of the biosafety cabinet. The first cavity is located on the side of the first piston 3 near the first end wall of the tube body, and the second cavity is located on the side of the first piston 3 away from the first end wall of the tube body.

[0087] Before the operator cleans and shreds the bile duct cancer tissue, the operator's forearm is inserted into the biosafety cabinet through the operating port. The operator then pulls down the fixing part and the lower end of the pull rope 4, and fixes the fixing part to the front end of the forearm. At this time, the pull rope 4 is in a taut state, and there is still a certain distance between the first piston 3 and the first end wall of the tube. The fixing part is located directly below the through hole.

[0088] The operator then cleans and shreds the bile duct cancer tissue. During these processes, the operator's forearm moves downwards, horizontally away from the axis of the through-hole, and horizontally closer to the axis of the through-hole.

[0089] When the operator's forearm moves downwards or horizontally away from the axis of the through hole, it pulls the lower end of the pull rope 4, causing the upper end of the pull rope 4 to pull the first piston 3 towards the first end wall of the tube. During the movement of the first piston 3 towards the first end wall of the tube, the liquid in the first cavity is forced into the second cavity through the first connecting hole. The faster the first piston 3 moves towards the first end wall of the tube, the greater the resistance it experiences. Therefore, the faster the fixed part and the pull rope 4 move, the greater the pulling force on the operator's forearm. When the speed at which the operator's forearm moves downwards or horizontally away from the axis of the through hole reaches a certain point, it cannot move any faster. The speed at which the operator's forearm moves downwards or horizontally away from the axis of the through hole is limited.

[0090] When the operator's forearm moves horizontally towards the axis of the through hole, the drive unit drives the first piston 3 to move away from the first end wall of the tube. During the movement of the first piston 3 away from the first end wall of the tube, the liquid in the second cavity is squeezed into the first cavity through the first connecting hole. The faster the first piston 3 moves away from the first end wall of the tube, the greater the resistance experienced by the first piston 3, and the greater the resistance experienced by the drive unit. When the speed at which the drive unit drives the first piston 3 to move away from the first end wall of the tube reaches a certain level, the drive unit can no longer drive the first piston 3 to move faster. The speed at which the drive unit drives the first piston 3 to move away from the first end wall of the tube is limited, and the speed at which the pull rope 4 moves toward the second cavity is also limited.

[0091] As the operator's forearm moves horizontally towards the axis of the through hole, the operator can observe whether the pull rope 4 is taut. If the pull rope 4 is slack, it indicates that the operator's forearm is moving too fast towards the axis of the through hole. By referring to the tension of the pull rope 4, the operator can control the movement speed of the forearm tip, simply by keeping the pull rope 4 taut. In this way, the speed at which the operator's forearm moves horizontally towards the axis of the through hole can be limited.

[0092] The first speed limiting unit can thus limit the speed of the operator's forearm in the horizontal direction and the speed of its downward movement, which is convenient when limiting the speed of the operator's forearm in the horizontal direction and the speed of its downward movement.

[0093] In this embodiment, the fixing part includes a fixing block 5, a strap 6, a hook and loop fastener 7, and a hook and loop fastener 8. The fixing block 5 is fixed to the lower end of the pull rope 4, the middle part of the strap 6 is fixed to the fixing block 5, the hook and loop fastener 7 is fixed to one end of the strap 6, and the hook and loop fastener 8 is located at the other end of the strap 6.

[0094] The operator wraps the strap 6 around the front end of the forearm, and then uses the hook and loop fasteners 7 and 8 to fix the two ends of the strap 6 together. At this time, the strap 6 can be fixed to the front end of the operator's arm, and the fixing part can be fixed to the front end of the operator's forearm.

[0095] In this embodiment, the wall of the perforation is recessed to form a first annular sealing groove. A first sealing ring 9 is provided inside the first sealing groove, and the first sealing ring 9 is press-fitted with the first sealing groove. The pull rope 4 is press-fitted with the inner hole of the first sealing ring 9. The first sealing ring 9 can play a sealing role, reducing the possibility of liquid in the first cavity leaking out through the gap between the pull rope 4 and the wall of the perforation.

[0096] In this embodiment, the tube includes a horizontal section 10, a curved section 11, and a vertical section 12. The horizontal section 10 is parallel to the front sidewall of the biosafety cabinet. The horizontal section 10 is fixedly connected to the fixing plate 1. The first end of the horizontal section 10 is the first end of the tube. The upper end of the curved section 11 is fixedly connected to the second end of the horizontal section 10. The upper end of the vertical section 12 is fixedly connected to the lower end of the curved section 11. The lower end of the vertical section 12 is the second end of the tube. The first piston 3 is slidably fitted within the horizontal section 10. The first speed limiting part also includes a first directional wheel 13 and a second directional wheel 14. Both the first directional wheel 13 and the second directional wheel 14 are rotatably connected to the fixed plate 1. The rotation center line of the first directional wheel 13 is set along the left and right direction, and the rotation center line of the second directional wheel 14 is set along the vertical direction. The drive unit includes a connecting rope 15 and a counterweight ball 16. One end of the connecting rope 15 is fixedly connected to the first piston 3, and the other end passes through the curved section 11 and is located at the lower end of the vertical section 12. The counterweight ball 16 is located inside the vertical section 12 and is fixedly connected to the other end of the connecting rope 15. The upper end of the pull rope 4 passes sequentially around the rear side of the first deflector wheel 13 and the front side of the second deflector wheel 14 before passing through the hole.

[0097] In the initial state, the counterweight ball 16 is supported on the lower end wall of the vertical section 12, the first piston 3 is located at the end of the straight section near the curved section 11, and the connecting rope 15 is in a taut state.

[0098] The first deflector wheel 13 and the second deflector wheel 14 can change the direction of the pull rope 4. After the upper end of the pull rope 4 passes around the rear side of the first deflector wheel 13 and the front side of the second deflector wheel 14 in sequence, the upper end of the pull rope 4 can be in a horizontal state and can be horizontally inserted into the hole.

[0099] As the operator's forearm moves downwards or horizontally away from the axis of the through hole, the piston moves toward the first end wall of the straight section. The upper end of the connecting rope 15 moves with the first piston 3 toward the first end wall of the straight section, and the lower end of the connecting rope 15 pulls the counterweight ball 16 upwards. After the piston moves a certain distance toward the first end wall of the straight section, the counterweight ball 16 can move upwards a certain distance.

[0100] As the operator's forearm moves horizontally towards the axis of the through hole, the tension on the first piston 3 towards the first end wall of the straight section decreases. The tension formed by the weight of the counterweight ball 16 on the connecting rope 15 can pull the first piston 3 away from the first end wall of the straight section. The drive unit can thus drive the first piston 3 away from the first end wall of the tube as the patient's forearm moves towards the axis of the through hole. This allows for relatively convenient driving of the first piston 3 away from the first end wall of the tube.

[0101] In this embodiment, the first steering wheel 13 has a first shaft hole, and the second steering wheel 14 has a second shaft hole. A first bracket 17 and a second bracket 18 are fixedly mounted on the fixed plate 1. The first bracket 17 has a first shaft 19, which rotatably engages with the first shaft hole. The second bracket 18 has a second shaft 20, which rotatably engages with the second shaft hole. Thus, the first steering wheel 13 and the second steering wheel 14 are rotatably connected to the fixed plate 1.

[0102] In this embodiment, the lower end of the through-hole wall and the lower side of the horizontal plate 2 form a rounded transition. This reduces the resistance to the pull rope 4 caused by the connection between the hole wall and the lower side of the horizontal plate 2 as the operator's forearm moves horizontally away from the axis of the through-hole.

[0103] In this embodiment, the second speed limiting part includes a vertical tube 21, a second piston 22, a lifting rod 23, and a pressure rod 24. The vertical tube 21 is fixedly connected to the fixing plate 1. Specifically, the vertical tube 21 is fixedly connected to the fixing plate 1 through a connecting rod 25. Both the upper and lower ends of the vertical tube 21 are closed. A circular hole is formed on the lower end wall of the vertical tube 21. The second piston 22 is slidably fitted inside the vertical tube 21. The second piston 22 divides the inner cavity of the vertical tube 21 into an upper cavity and a lower cavity. Both the upper cavity and the lower cavity are filled with liquid. A second connecting hole is formed on the second piston 22 to connect the upper cavity and the lower cavity. The upper end of the lifting rod 23 passes through the circular hole into the lower cavity and connects to the second piston 22. The lifting rod 23 is slidably fitted inside the circular hole. The pressure rod 24 is fixed to the lower end of the lifting rod 23 and located on the rear side of the fixing part. The pressure rod 24 is arranged in the left-right direction.

[0104] In the initial state, the pressure bar 24 rests against the work surface of the biosafety cabinet.

[0105] Before fixing the fixing part to the front end of the operator's forearm, push the pressure rod 24, the lifting rod 23 and the second piston 22 upward. After fixing the fixing part to the front end of the operator's forearm, the pressure rod 24 can be pressed on the operator's forearm and located behind the fixing part.

[0106] During the process of cleaning and dissecting the bile duct cancer tissue, the operator's forearm will move upwards.

[0107] During the upward movement of the operator's forearm, the forearm can push the pressure rod 24, the lifting rod 23, and the second piston 22 upwards. The liquid in the upper cavity is squeezed into the lower cavity through the second connecting hole. The faster the second piston 22 moves upwards, the greater the resistance it experiences. Therefore, the faster the front part of the operator's forearm moves upwards, the greater the resistance it experiences. When the operator's forearm reaches a certain upward speed, it cannot move any faster. The upward speed of the operator's forearm is limited.

[0108] The second speed limiter thus restricts the speed at which the operator's forearm moves upward. This is convenient for limiting the speed at which the operator's forearm moves upward.

[0109] As the operator's forearm moves downward, the pressure rod 24, the lifting rod 23, and the second piston 22 can move downward under their own gravity, and the pressure rod 24 can hold the operator's forearm.

[0110] In this embodiment, the wall of the circular hole is recessed to form a second annular sealing groove. A second sealing ring 26 is provided inside the second sealing groove. The second sealing ring 26 is interference-fitted with the second sealing groove, and the lifting rod 23 is interference-fitted with the inner hole of the second sealing ring 26. The second sealing ring 26 can play a sealing role, reducing the possibility of liquid in the lower cavity leaking out through the gap between the lifting rod 23 and the wall of the circular hole.

[0111] One of the biosafety cabinet auxiliary devices can only limit the movement speed of one of the operator's forearms inside the biosafety cabinet. If it is necessary to limit the movement speed of both of the operator's forearms inside the biosafety cabinet, it is only necessary to install two of the biosafety cabinet auxiliary devices inside the biosafety cabinet.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for culturing bile duct carcinoma organoids, characterized in that, Includes the following steps: S1. Clean the bile duct cancer tissue in a biosafety cabinet and then chop the bile duct cancer tissue into small pieces; S2. Transfer the chopped tissue to a centrifuge tube containing tissue digestion solution for digestion, and collect the supernatant suspension. S3. After centrifuging the collected upper suspension, collect the cholangiocarcinoma tissue cell precipitate. S4. Inoculate cholangiocarcinoma tissue cells into matrix gel. S5. After the matrix gel solidifies, culture the tissue cells using the corresponding organoid culture medium to obtain primary organoids. Change the culture medium every 3 days.

2. The method for culturing bile duct carcinoma organoids according to claim 1, characterized in that, Step S5 is followed by the following steps: S6. When the density of primary organoids reaches 80%-90% of the matrix gel space, add ice-cooled DMEM washing medium, and use a cold pipette tip to dissociate the matrix gel, or place the 24-well plate in a -20°C freezer for 4 minutes to aid in matrix gel dissociation. Then collect the mixture in the wells into a 15 ml centrifuge tube, add 2-3 times the volume of ice-cooled DMEM washing medium to the centrifuge tube to dilute the matrix gel and organoid mixture, and centrifuge the mixture at 4°C and 300×g for 5 min. S7. After centrifugation, remove the supernatant, add 1 ml TrypLE Express enzyme to the organoid precipitate, and incubate the mixture in a rotary thermostat at 37°C and 30 rpm for 10 min. Add 10 ml of ice-cooled Advanced DMEM / F-12 washing medium to stop digestion, and centrifuge the mixture at 4°C and 300×g for 5 min. S8. After centrifugation, observe whether the organoids have completely precipitated and whether there is any unremoved matrix gel-primary organoid mixture in the intermediate layer. If only obvious cells or cell clusters are observed to precipitate, remove the supernatant, add cold, fresh Cultrex Reduced Growth Factor Basement Membrane Extract (type 2) to resuspend the precipitate, and re-inoculate at a ratio of 1:2 to 1:

4. Use a pipette to add the precipitate to each well of a 24-well plate. Invert the 24-well plate and incubate it in a sterile cell culture incubator at 37°C with 5% CO2 for about 30 minutes until it solidifies into a dome shape. After the Basement Membrane Extract has solidified, add cholangiocarcinoma organoid culture medium for culturing; or add organoid cryopreservation solution to the precipitate and freeze it in a liquid nitrogen tank for long-term storage.

3. The method for culturing bile duct carcinoma organoids according to claim 1, characterized in that, The tissue cleaning process in step S1 includes the following steps: S11. Place the obtained cholangiocarcinoma tissue into a 10cm cell culture dish; S12. Add 10 ml of DMEM washing medium containing 1% FBS, 1% Penicillin-Streptomycin-Neomycin, and 100 µg / ml Primocin to the culture dish to wash the tissue.

4. The method for culturing bile duct carcinoma organoids according to claim 1, characterized in that: The tissue digestion solution in step S2 comprises the following components: based on Advanced DMEM / F-12 medium, with the following final concentrations added: 10 mM HEPEs, 1x GlutaMAX Supplement, 100 µg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin, 2.5% FBS, 5 mg / ml Collagenase II, 1 mg / ml Dispase II, 10.5 µM Y-27632, and 10 µg / ml DNAse I.

5. The method for culturing bile duct carcinoma organoids according to claim 1, characterized in that: The cleaning and shredding of bile duct cancer tissue in the biosafety cabinet are performed with the assistance of a biosafety cabinet auxiliary device used to limit the speed of the operator's forearm movement within the biosafety cabinet.

6. The method for culturing bile duct carcinoma organoids according to claim 5, characterized in that: The biosafety cabinet uses an auxiliary device including a fixing plate, a first speed limiting part, and a second speed limiting part. The fixing plate is used to fix the device to the front side wall of the biosafety cabinet. The first speed limiting part and the second speed limiting part are both located inside the biosafety cabinet and are disposed on the fixing plate. The first speed limiting part is used to fix and connect the front end of the operator's forearm and to limit the speed of the operator's forearm moving horizontally and downward. The second speed limiting part is used to press down against the operator's forearm and to limit the speed of the operator's forearm moving upward.

7. The method for culturing bile duct carcinoma organoids according to claim 6, characterized in that: The first speed limiting part includes a horizontal plate, a tube, a first piston, a pull rope, a fixing part, and a driving part. The front end of the horizontal plate is fixed on the horizontal plate, and the rear end extends horizontally backward. A through hole is formed at the rear end of the horizontal plate. The first end and the second end of the tube are both closed. A perforation is formed on the end wall of the first end of the tube. The first piston slides in the tube and divides the inner cavity of the tube into a first cavity and a second cavity. Both the first cavity and the second cavity are filled with liquid. A first connecting hole is formed on the first piston to connect the first cavity and the second cavity. The lower end of the pull rope passes downward through the through hole, and the upper end of the pull rope passes through the perforation into the first cavity and is fixedly connected to the first piston. The pull rope slides in the perforation. The fixing part is fixedly connected to the lower end of the pull rope and is used to fix it to the front end of the operator's forearm. The driving part is used to drive the first piston to move away from the first end wall of the tube during the movement of the patient's forearm end near the axis of the through hole.

8. The method for culturing bile duct carcinoma organoids according to claim 7, characterized in that: The tube body includes a horizontal section, a curved section, and a vertical section. The horizontal section is parallel to the front side wall of the biosafety cabinet and is fixedly connected to the fixing plate. The first end of the horizontal section is the first end of the tube body. The upper end of the curved section is fixedly connected to the second end of the horizontal section. The upper end of the vertical section is fixedly connected to the lower end of the curved section. The lower end of the vertical section is the second end of the tube body. The first piston is slidably fitted within the horizontal section. The first speed limiting part also includes a first steering wheel and a second steering wheel. Both the first steering wheel and the second steering wheel are rotatably connected to the fixed plate. The rotation center line of the first steering wheel is set along the left and right direction, and the rotation center line of the second steering wheel is set along the vertical direction. The drive unit includes a connecting rope and a counterweight ball. One end of the connecting rope is fixedly connected to the first piston, and the other end passes through the curved section and is located at the lower end of the vertical section. The counterweight ball is located inside the vertical section and is fixedly connected to the other end of the connecting rope. The upper end of the pull rope passes sequentially around the rear side of the first deflector wheel and the front side of the second deflector wheel before passing through the hole.

9. The method for culturing bile duct carcinoma organoids according to claim 7, characterized in that: The lower end of the through hole wall and the lower side of the horizontal plate form a rounded transition.

10. The method for culturing bile duct carcinoma organoids according to claim 6, characterized in that: The second speed limiting part includes a vertical tube, a second piston, a lifting rod, and a pressure rod. The vertical tube is fixedly connected to the fixing plate, and both the upper and lower ends of the vertical tube are closed. A circular hole is formed on the lower end wall of the vertical tube. The second piston is slidably fitted inside the vertical tube, dividing the inner cavity of the vertical tube into an upper cavity and a lower cavity. Both the upper cavity and the lower cavity are filled with liquid. A second connecting hole is formed on the second piston to connect the upper cavity and the lower cavity. The upper end of the lifting rod passes through the circular hole into the lower cavity and connects to the second piston. The lifting rod is slidably fitted inside the circular hole. The pressure rod is fixed to the lower end of the lifting rod and located on the rear side of the fixing part. The pressure rod is arranged in the left-right direction.

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