Method for culturing bile duct organoid based on postoperative bile drainage

Abstract

The invention relates to the technical field of bile duct type organ culture, and particularly discloses a method for culturing a bile duct type organ based on postoperative drainage bile, which comprises the following steps: collecting bile, detecting cell survival rate, filtering, enriching stem cells by a multi-step centrifugation method, culturing the bile duct type organ and the like. The problems that direct surgical resection or biopsy specimen acquisition is limited, repeated sampling cannot be carried out for multiple times, and potential ethical disputes are avoided. By optimizing bile sampling and treatment processes, the method effectively solves the problem that excessive digestion or insufficient digestion is easy to occur when primary tissues are digested by traditional digestive enzymes, so that the purity and activity of bile duct cell separation are improved, the success rate of organoid culture is further improved, meanwhile, the operation steps are more concise and clear, and the method is suitable for large-scale popularization and application. And the technical requirements on operators are reduced, so that green hands can quickly grasp and successfully implement, and wide popularization and application of the technology are facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of bile duct organoid culture, and specifically relates to a method for culturing bile duct organoids based on postoperative drainage bile. Background Art

[0002] Biliary organoids, also known as biliary epithelial cell organoids, are three-dimensional cellular structures formed from bile duct-related stem or progenitor cells through induced differentiation and self-assembly. These tiny tissue mimics have characteristic markers of bile duct cells and are highly similar to the in vivo bile duct in both morphology and function. The formation process of bile duct organoids reflects the self-renewal capacity and tissue-specific differentiation potential of cells, making them ideal biological tools for studying bile duct physiology and pathology.

[0003] Currently, liver, gallbladder, and bile duct tissues are dissociated into single cells (primary bile duct cells are obtained from biopsy or surgically removed tissues by two-step collagenase perfusion digestion), and bile duct cells with stem cell differentiation potential are further enriched by sedimentation and multi-step centrifugation, and then embedded in matrix gel and cultured into three-dimensional spherical bile duct organoids in a specific culture medium. During ERCP or PTCD surgery, bile is drained from the bile duct through a catheter to culture organoids. Biliary epithelial cells are directly collected by endoscopic common bile duct cell brushing under ERCP and cultured organoids.

[0004] Clinically, liver, gallbladder or bile duct tissues are usually obtained through surgical resection or biopsy. The specimens need to be taken at the time of surgery and cannot be sampled repeatedly. There are many restrictions and there may be ethical and regulatory issues. The key step in the existing technology to separate bile duct cells from liver or gallbladder tissue is to add digestive enzymes to digest the primary tissue. This step is prone to over- or under-digestion. Over-digestion affects the clustering of stem cells, and under-digestion reduces the number of bile duct cells in the final precipitate, both of which will reduce the success rate of organoid culture. During ERCP or PTCD surgery, bile is drained from the bile duct through a catheter, and the amount of bile drainage is small (only 2-5mL). The endoscopic common bile duct cell brushing procedure through ERCP has high requirements on personal skills and is complex and difficult to sample.

[0005] Both of the above operations have risks such as bile duct infection and direct damage to the bile duct wall, and are not suitable for novices.

[0006] In summary, existing technologies have many shortcomings in obtaining and processing liver, gallbladder or bile duct tissue samples, including strict restrictions on ethical supervision, instability of technical operations, and limitations and risks of clinical sampling methods. Therefore, it is urgent to develop a method for culturing bile duct organoids based on postoperative drainage bile to overcome the above difficulties. Summary of the invention

[0007] The object of the present invention is to provide a method for culturing bile duct organoids based on postoperative drainage bile to solve the problems raised in the above-mentioned background technology.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A method for culturing bile duct organoids based on postoperative drainage bile, comprising the following steps:

[0010] S1. Collecting bile:

[0011] Collect fresh bile from the drainage tube or negative pressure bag of ERCP or PTCD patients in a sterile bottle with buffer added, place the sterile bottle on ice and transfer it to a biosafety cabinet;

[0012] The buffer contains DMEM / F-12 medium containing 1% volume of L-alanyl-L-glutamine (GlutaMax, 100×), 1% volume of HEPES buffer and 3% volume of Promicin (antibiotic solution);

[0013] S11. Detection of cell survival rate:

[0014] Shake and mix the bile, pipette 20 μL of bile into a 0.2 mL EP tube, add an equal volume of AOPI fluorescent dye, mix well, and use a cell detector to detect cell viability;

[0015] S2. Filtration and multi-step centrifugation to enrich stem cells:

[0016] S21, prepare the tips of various ranges, 50mL centrifuge tubes, 100μm filter screens, 0.2mL EP tubes and 1.5mL EP tubes, and rinse them with a rinse solution before use; the rinse solution is PBS containing 7.5% BSA;

[0017] S22. Add 5 times the volume of buffer to the sterile bottle containing fresh bile, shake and mix, then filter the mixture through a 100 μm diameter filter into a 50 mL centrifuge tube to remove impurities such as cell debris.

[0018] S23, place the centrifuge tube in the previous step in a 4°C centrifuge at 300 g for 5 minutes to collect the precipitate;

[0019] S24, cracking red treatment:

[0020] Because some bile contains red blood cells, the precipitate in S23 needs to be lysed before proceeding to the next step to remove the red blood cells in the bile; the specific steps are to add red blood cell lysis solution to the precipitate collected after centrifugation of S23, and perform lysis reaction at 4°C for 10 minutes, shaking once every 2 minutes during the reaction, and adding a buffer twice the volume of the red blood cell lysis solution after 10 minutes to terminate the reaction, and centrifuge at 300g in a 4° centrifuge for 5 minutes;

[0021] S25, resuspend the cell pellet at the bottom of the 50 mL centrifuge tube with 1 mL of buffer, collect it into a 1.5 mL EP tube, and centrifuge it at 300 g in a 4° centrifuge for 5 minutes;

[0022] S26, discard the supernatant in the 1.5 mL EP tube after centrifugation in S25, resuspend the precipitate with 1 mL buffer, and repeat the centrifugation procedure in S25;

[0023] S27, discard the supernatant in the 1.5mL EP tube after centrifugation in S26, resuspend the precipitate with 1mL buffer, pipette 20μL of the resuspended solution into a 0.2mL EP tube, add an equal volume of AOPI fluorescent dye, mix well, and use a cell detector to detect the cell number and viability; place the 1.5mL centrifuge tube in a 4° centrifuge again and centrifuge at 300g for 5 minutes;

[0024] S28. Discard the supernatant in the 1.5 mL EP tube after centrifugation in S27, collect the precipitate, and count the number of cells in the precipitate.

[0025] S3. Cultivation of bile duct organoids

[0026] S31, add a certain amount of liquid matrix gel at a ratio of 50 μL matrix gel per 100,000 cells, pipette and mix well, and drop it in the center of the TC-treated 24-well cell culture plate, place the cell culture plate in a 37°C cell culture incubator with 5% carbon dioxide, and incubate for at least 20 minutes to wait for the matrix gel to solidify;

[0027] S32. Add 500 μl of serum-free medium for organoids to each well. Add 1 / 1000 volume of Y27632 (anti-apoptotic factor) and 1 / 500 volume of Primocin to the medium. Change the medium every 2-3 days and continue to observe and record under an inverted microscope.

[0028] Preferably, the buffer in S1, S24, S25, S26 and S27 comprises 1% volume of L-alanyl-L-glutamine (GlutaMax, 100×), 1% volume of HEPES buffer and 3% volume of DMEM / F-12 culture medium containing penicillin-streptomycin double antibody solution.

[0029] Preferably, the volume ratio of bile to buffer in S1 is 1:5; the fresh bile is bile drawn from the drainage tube within 1 hour after flowing into the negative pressure bag or immediately.

[0030] Preferably, the rinse solution in step S21 is PBS containing 7.5% BSA.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The invention uses postoperative drainage bile as a sample source, avoiding the ethical controversy and regulatory restrictions brought about by direct surgical resection or biopsy, and is easier to obtain approval from regulatory agencies, and can be sampled repeatedly. By optimizing the bile sampling and processing process, the present invention effectively solves the problem of over- or under-digestion that is prone to occur when traditional digestive enzymes digest primary tissues, thereby improving the purity and activity of bile duct cell isolation, and further improving the success rate of organoid culture. By utilizing postoperative drainage bile, the present invention avoids the sampling volume restrictions of bile drainage via catheter during ERCP or PTCD surgery, as well as the high technical requirements and complexity of common bile duct cell brushing, significantly reducing the risks of bile duct infection, direct damage to the bile duct wall, etc., making the sampling process safer and simpler.

[0033] At the same time, the present invention improves the culture efficiency and success rate. Compared with the traditional method, the operation steps of the present invention are more concise and clear, and the technical requirements for the operator are reduced, so that novices can quickly master and successfully implement it, which is conducive to the wide promotion and application of the technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a diagram showing that adding a buffer solution during sampling effectively improves cell survival rate in the present invention;

[0035] Figure 2 This is a comparison of the primary organoids of the present invention on day 1, day 4, and day 7;

[0036] Figure 3 This is an observation diagram showing that the bile-derived bile duct organoids of the inventors can be stably passaged;

[0037] Figure 4 This is a comparison of the proliferation rates of organoids cultured from bile and from surgically derived liver tissues by the inventor;

[0038] Figure 5 This is a diagram of the organoid of the present invention expanding from 300 μm on day 4 to 500 μm on day 7;

[0039] Figure 6 This is the H&E staining picture of the organoid of the present invention;

[0040] Figure 7 This is a positive staining image of CK19, a specific marker for bile duct epithelium in the organoid of the present invention. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Embodiment 1:

[0043] See also Figure 1-7 As shown, a method for culturing bile duct organoids based on postoperative drainage bile comprises the following steps:

[0044] S1. Collecting bile:

[0045] Collect fresh bile from the drainage tube or negative pressure bag of ERCP or PTCD patients in a sterile bottle with buffer added, place the sterile bottle on ice and transfer it to a biosafety cabinet;

[0046] The buffer contains DMEM / F-12 medium containing 1% volume of L-alanyl-L-glutamine (GlutaMax, 100×), 1% volume of HEPES buffer and 3% volume of Promicin (antibiotic solution);

[0047] During this period, the bile sampling process required the protection of buffer (5 times the volume of bile + 30u of Y-27632). We tested the bile samples in two sterile bottles with and without buffer overnight (12h). We then compared the cell counts (10μL bile + 10μL AOPI solution mixture). Figure 1 , the cell count of bile samples without buffer was almost 0, in sharp contrast to that with buffer.

[0048] S11. Detection of cell survival rate:

[0049] Shake and mix the bile, pipette 20 μL of bile into a 0.2 mL EP tube, add an equal volume of AOPI fluorescent dye, mix well, and use a cell detector to detect cell viability;

[0050] S2. Filtration and multi-step centrifugation to enrich stem cells:

[0051] S21, prepare the tips of various ranges, 50mL centrifuge tubes, 100μm filter screens, 0.2mL EP tubes and 1.5mL EP tubes, and rinse them with a rinse solution before use; the rinse solution is PBS containing 7.5% BSA;

[0052] S22. Add 5 times the volume of buffer to the sterile bottle containing fresh bile, shake and mix, then filter the mixture through a 100 μm diameter filter into a 50 mL centrifuge tube to remove impurities such as cell debris.

[0053] S23, place the centrifuge tube in the previous step in a 4°C centrifuge at 300 g for 5 minutes to collect the precipitate;

[0054] S24, cracking red treatment:

[0055] Because some bile contains red blood cells, the precipitate in S23 needs to be lysed before proceeding to the next step to remove the red blood cells in the bile; the specific steps are to add red blood cell lysis solution to the precipitate collected after centrifugation of S23, and perform lysis reaction at 4°C for 10 minutes, shaking once every 2 minutes during the reaction, and adding a buffer twice the volume of the red blood cell lysis solution after 10 minutes to terminate the reaction, and centrifuge at 300g in a 4° centrifuge for 5 minutes;

[0056] S25, resuspend the cell pellet at the bottom of the 50 mL centrifuge tube with 1 mL of buffer, collect it into a 1.5 mL EP tube, and centrifuge it at 300 g in a 4° centrifuge for 5 minutes;

[0057] S26, discard the supernatant in the 1.5 mL EP tube after centrifugation in S25, resuspend the precipitate with 1 mL buffer, and repeat the centrifugation procedure in S25;

[0058] S27, discard the supernatant in the 1.5mL EP tube after centrifugation in S26, resuspend the precipitate with 1mL buffer, pipette 20μL of the resuspended solution into a 0.2mL EP tube, add an equal volume of AOPI fluorescent dye, mix well, and use a cell detector to detect the cell number and viability; place the 1.5mL centrifuge tube in a 4° centrifuge again and centrifuge at 300g for 5 minutes;

[0059] S28. Discard the supernatant in the 1.5 mL EP tube after centrifugation in S27, collect the precipitate, and count the number of cells in the precipitate.

[0060] S3. Cultivation of bile duct organoids

[0061] S31, add a certain amount of liquid matrix gel at a ratio of 50 μL matrix gel per 100,000 cells, pipette and mix well, and drop it in the center of the TC-treated 24-well cell culture plate, place the cell culture plate in a 37°C cell culture incubator with 5% carbon dioxide, and incubate for at least 20 minutes to wait for the matrix gel to solidify;

[0062] S32. Add 500 μl of serum-free medium for organoids to each well. Add 1 / 1000 volume of Y27632 (anti-apoptotic factor) and 1 / 500 volume of Primocin to the medium. Change the medium every 2-3 days and continue to observe and record under an inverted microscope.

[0063] The buffer in S1, S24, S25, S26 and S27 comprises 1% L-alanyl-L-glutamine (GlutaMax, 100×), 1% HEPES buffer and 3% penicillin-streptomycin dual antibody solution in DMEM / F-12 medium. The volume ratio of bile to buffer in S1 is 1:5; the fresh bile is the bile drawn from the drainage tube within 1 hour of flowing into the negative pressure bag or immediately, and the rinse solution in step S21 is PBS containing 7.5% BSA.

[0064] The above instruments and reagents include: 50 mL centrifuge tubes (Corning, America), pipettes (Eppendorf, Germany), 100 m filter (Corning, America), inverted microscope (Nikon, Japan), pipette tips of various ranges (Bioland, China), centrifuge (Cenlee, China), TC-treated 24-well cell culture plates (Corning, America), cell counter (Countstar, China), and cell counting chamber (Countstar, China). DMEM / F12 (Gibco, America), PBS (Biosharp, America), HEPES (Life Science product Services), GultaMax (Gibico, America), penicillin-streptomycin double antibody solution (Gibico, America), BSA (MCE, America), Matrigel (Corning, America), AOPI cell fluorescent dye (Countstar, China), Primocin (Invivogen), Y27632 (MCE, America).

[0065] Reference column, morphological observation of bile organoids cultured from primary biliary epithelial cells isolated from bile.

[0066] The growth process and morphological changes of bile duct organoids were observed under an inverted microscope (from left to right, primary organoids on day 1, day 4, and day 7). It can be seen that 24 hours after implantation (day 1), some bile duct epithelial cells have differentiated into bile duct organoids with vacuolar structures. After 96 hours (day 4), they gradually expanded into vesicular organoids with larger diameters. After one week of culture, they grew into vacuolar organoids with diameters of 50 to 200 μm. Figure 2 shown.

[0067] Human bile-derived bile duct organoids cultured using the method of the present invention can be stably passaged ( Figure 3 ):

[0068] Around the 10th day, the P0 bile duct organoids were subcultured, and the growth process and morphological changes of the P1 bile duct organoids were continuously observed under an inverted microscope (from left to right, the 1st, 3rd, 5th and 7th days of the P1 generation). It can be seen under the inverted microscope that the P1 bile duct organoids are cystic, plump and translucent, and expand slightly slower than the original generation.

[0069] We also compared bile duct organoids cultured from human bile with those from surgically derived liver tissue ( Figure 4 ), starting from the third day of culture (72h), we took pictures of the bile duct organoids from both sources every 2 hours for 3 days. It can be seen that the growth rate of bile duct organoids from bile is better than that from liver throughout the process, and the proliferation rate is faster. We recorded the expansion of the organoids for three days. Its diameter expanded from 300μm at the beginning to 500μm. Figure 5 We sent the cultured bile duct organoids for H&E pathological staining ( Figure 6 ), bile duct cytokeratin CK19 can specifically stain the bile duct epithelium, and the organoids submitted for examination were positive for CK19 staining ( Figure 7 ).

[0070] It will be appreciated that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will be a routine task of design, fabrication, and production for those of ordinary skill having the benefit of this disclosure without undue experimentation.

[0071] The present invention optimizes the bile sampling process, optimizes the process of culturing bile duct organoids, improves the storage of primary samples, and the methods of culturing and reducing cell loss. At the same time, the culturing method is simpler, suitable for novice operation, and the sampling is safer, avoiding possible infection to patients caused by intraoperative sampling, and improving the source and safety of sampling.

[0072] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for culturing bile duct organoids based on postoperative drainage bile, characterized in that: The following steps are involved: S1. Collecting bile: Collect fresh bile from the drainage tube or negative pressure bag of ERCP or PTCD patients in a sterile bottle with buffer added, place the sterile bottle on ice and transfer it to a biosafety cabinet; S11. Detection of cell survival rate: Shake and mix the bile, pipette 20 μL of bile into a 0.2 mL EP tube, add an equal volume of AOPI fluorescent dye, mix well, and use a cell detector to detect cell viability; S2. Filtration and multi-step centrifugation to enrich stem cells: S21. Prepare the pipette tips of various ranges, 50 mL centrifuge tubes, 100 μm filter screens, 0.2 mL EP tubes and 1.5 mL EP tubes, and rinse them with rinse solution before use; S22, add 5 times the volume of buffer to the sterile bottle containing fresh bile, shake and mix, and then filter the mixture through a 100m diameter filter into a 50mL centrifuge tube to remove impurities such as cell debris; S23, place the centrifuge tube in the previous step in a 4°C centrifuge at 300 g for 5 minutes to collect the precipitate; S24, cracking red treatment: Because some bile contains red blood cells, the precipitate in S23 needs to be lysed before proceeding to the next step to remove the red blood cells in the bile; the specific steps are to add red blood cell lysis solution to the precipitate collected after centrifugation of S23, and perform lysis reaction at 4°C for 10 minutes, shaking once every 2 minutes during the reaction, and adding a buffer twice the volume of the red blood cell lysis solution after 10 minutes to terminate the reaction, and centrifuging at 300g in a 4°C centrifuge for 5 minutes; S25, resuspend the cell pellet at the bottom of the 50 mL centrifuge tube in S24 with 1 mL of buffer, collect into a 1.5 mL EP tube, and centrifuge at 300 g in a 4 °C centrifuge for 5 min; S26, discard the supernatant in the 1.5 mL EP tube after centrifugation in S25, resuspend the precipitate with 1 mL buffer, and repeat the centrifugation procedure in S25; S27, discard the supernatant in the 1.5mL EP tube after centrifugation in S26, resuspend the precipitate with 1mL buffer, pipette 20μL of the resuspended solution into a 0.2mL EP tube, add an equal volume of AOPI fluorescent dye, mix well, and use a cell detector to detect the cell number and viability; place the 1.5mL centrifuge tube in a 4℃ centrifuge again and centrifuge at 300g for 5 minutes; S28. Discard the supernatant in the 1.5 mL EP tube after centrifugation in S27, collect the precipitate, and count the number of cells in the precipitate. S3. Cultivation of bile duct organoids S31, add a certain amount of liquid matrix gel at a ratio of 50 μL matrix gel per 100,000 cells, pipette and mix well, and drop it in the center of the TC-treated 24-well cell culture plate, place the cell culture plate in a 37°C cell culture incubator with 5% carbon dioxide, and incubate for at least 20 minutes to wait for the matrix gel to solidify; S32. Add 500 μL of serum-free medium for organoids to each well. Add 1 / 1000 volume of Y27632 (anti-apoptotic factor) and 1 / 500 volume of Primocin to the medium. Change the medium every 2-3 days and continue to observe and record under an inverted microscope.

2. A method for culturing bile duct organoids based on postoperative drainage bile according to claim 1, characterized in that: The buffer in S1, S24, S25, S26 and S27 contains 1% volume of L-alanyl-L-glutamine (GlutaMax, 100×), 1% volume of HEPES buffer and 3% volume of DMEM / F-12 culture medium containing penicillin-streptomycin double antibody solution.

3. The method for culturing bile duct organoids based on postoperative drainage bile according to claim 1, characterized in that: The volume ratio of bile to buffer in the S1 is 1:5; the fresh bile is the bile drawn from the drainage tube within 1 hour after flowing into the negative pressure bag or immediately.

4. The method for culturing bile duct organoids based on postoperative drainage bile according to claim 1, characterized in that: The rinse solution in step S21 is PBS containing 7.5% BSA.

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