Pancreatic cancer organoid passage method and application thereof

Abstract

The invention relates to a pancreatic cancer organ passage method and application thereof, and belongs to the technical field of biology. The invention provides a passage method of pancreatic cancer organoid. The passage method comprises a dispersion step, the dispersing step comprises the step of mechanically dispersing the pancreatic cancer organs needing passage through blowing and beating. Research finds that the organoid can achieve the dispersion effect almost the same as that of the organoid obtained by using enzyme digestion under the condition of not adding enzyme digestion and only depending on blowing and beating, and the organoid obtained by mechanical blowing and beating dispersion has a faster and better growth trend. Therefore, the passage method provided by the invention has the advantages of short-term rapid growth, economy and high efficiency when being used for passage of the organoid, the survival rate and growth efficiency of the organoid can be improved, the number and density of the organoid required by experiments can be quickly achieved, the in-vitro culture period of the organoid can be shortened, the culture efficiency of the organoid can be improved, and the culture cost of the organoid can be reduced. The experiment process is better accelerated, the waste of consumables such as reagents can be reduced, and the cost is effectively reduced.

Description

Technical Field

[0001] The invention relates to a pancreatic cancer organoid culture method and application thereof, and belongs to the field of biotechnology. Background Art

[0002] Pancreatic cancer organoids are miniature organs that are obtained by 3D culture of stem cells or progenitor cells from pancreatic cancer patients in vitro through cell self-renewal and self-assembly. They contain multiple cell types, perform certain specific functions, and form a spatial structure similar to organs in the body. Currently, pancreatic cancer organoids have been widely used in drug screening, new drug development, and disease model research.

[0003] During the in vitro culture of organoids, the sustainability, stability and efficiency of organoid expansion greatly affect the experimental process. Therefore, the selection and process of organoid passaging methods are particularly important. The traditional organoid passaging method is to release and dissociate organoids using TrypLE or a combination of TrypLE and Dispase. However, in the actual culture process, it was found that the dissociation of TrypLE would affect the formation rate of organoids, increase the difficulty of organoid formation, and cause a certain amount of organoid loss during the washing process of removing TrypLE, which would extend the growth cycle of the organoids. The extension of the experimental process will require more time, energy and reagents for maintenance by the culture personnel, thereby generating more economic costs. Therefore, there is a need for an economical and efficient passaging method that is conducive to the short-term rapid growth of pancreatic cancer organoids, so as to improve the survival rate and growth efficiency of organoids, shorten the in vitro culture cycle of organoids, improve the culture efficiency of organoids, reduce the waste of reagents and other consumables, and effectively reduce costs. Summary of the invention

[0004] To solve the above-mentioned defects, the present invention provides a method for passaging pancreatic cancer organoids, which comprises a dispersion step; the dispersion step comprises: mechanically dispersing the pancreatic cancer organoids to be passaged by blowing.

[0005] In one embodiment of the present invention, the dispersing step comprises: blowing the pancreatic cancer organoids to be passaged 10 to 30 times at 0 to 4° C. to mechanically disperse the pancreatic cancer organoids to be passaged.

[0006] In one embodiment of the present invention, the dispersion step comprises: after discarding the organoid culture medium in the pancreatic cancer organoid culture container, adding Organoid Wash precooled to 0-4°C to the pancreatic cancer organoid culture container to obtain an organoid suspension; after collecting the organoid suspension into a pancreatic cancer organoid dispersion container precooled to 0-4°C, blowing the organoid suspension 10-30 times at 0-4°C to mechanically disperse the pancreatic cancer organoids to be passaged to obtain a dispersed organoid suspension A; after centrifuging the dispersed organoid suspension A at 0-4°C, discarding the supernatant to obtain a pancreatic cancer organoid dispersion container containing organoid precipitate A; adding Organoid Wash precooled to 0-4°C to the pancreatic cancer organoid dispersion container to obtain the organoid suspension; After Wash is added to the pancreatic cancer organoid dispersion container containing organoid precipitate A, the organoid suspension is blown 10 to 30 times at 0 to 4°C to mechanically disperse the pancreatic cancer organoids to be passaged to obtain a dispersed organoid suspension B; after centrifuging the dispersed organoid suspension B at 0 to 4°C, the supernatant is discarded to obtain a pancreatic cancer organoid dispersion container containing organoid precipitate B.

[0007] In one embodiment of the present invention, the centrifugal speed is 1200-1500 rpm and the time is 5-10 min.

[0008] In one embodiment of the present invention, the subculture method also includes a subculture step; the subculture step is arranged after the dispersion step; the subculture step includes: adding matrix gel to a pancreatic cancer organoid dispersion container containing organoid precipitate B to obtain a mixture of organoids and matrix gel; adding the mixture of organoids and matrix gel to a pancreatic cancer organoid culture container, and after the mixture of organoid precipitate and matrix gel is solidified, adding organoid culture medium to the pancreatic cancer organoid culture container to subculture the organoids to obtain subcultured pancreatic cancer organoids.

[0009] In one embodiment of the present invention, in the passaging step, the passaging ratio of organoids is 2 to 3:1 (when passaging organoids, if the passaging ratio of organoids is 2:1, one organoid is divided into two equal parts according to the number for passaging; if the passaging ratio of organoids is 3:1, one organoid is divided into three equal parts according to the number for passaging).

[0010] In one embodiment of the present invention, the components of the Organoid Wash include N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), glutamine supplement (GlutaMAX Supplement), penicillin-streptomycin (Penicillin-Streptomycin), primary cell antibiotic Primocin, fetal bovine serum (BSA) and ROCK-I / II inhibitor Y-27632.

[0011] In one embodiment of the present invention, the concentration of the N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid in Organoid Wash is 8 to 12 mM; the concentration of the glutamine supplement in Organoid Wash is 1.8 to 2.2 mM; the concentration of penicillin-streptomycin in Organoid Wash is 80 to 120 U / mL; the concentration of the primary cell antibiotic Primocin in Organoid Wash is 80 to 120 μg / mL; in terms of volume percentage, the concentration of the fetal bovine serum in Organoid Wash is 0.08 to 0.12%; the concentration of the ROCK-I / II inhibitor Y-27632 in Organoid Wash is 8 to 12 μM.

[0012] In one embodiment of the present invention, the components of the Organoid Wash further include a cell culture medium; the cell culture medium includes DMEM basal culture medium, DMEM / F12 basal culture medium and / or Advanced DMEM / F-12 basal culture medium.

[0013] In one embodiment of the present invention, during the passaging step, the pancreatic cancer organoid culture container containing the organoid culture medium is preheated to 37°C.

[0014] The present invention also provides a method for screening drugs for treating pancreatic cancer, the method comprising: firstly using the above-mentioned passaging method to pass on pancreatic cancer organoids, and then using the pancreatic cancer organoids obtained by passaging as an in vitro model of pancreatic cancer to screen drugs for treating pancreatic cancer.

[0015] In one embodiment of the present invention, the method comprises: using the above-mentioned passaging method to pass the pancreatic cancer organoids to obtain an in vitro model of pancreatic cancer; administering the drug to be screened to the in vitro model of pancreatic cancer, and evaluating the therapeutic effect of the drug to be screened on pancreatic cancer based on changes in indicators of the in vitro model of pancreatic cancer, thereby obtaining a drug that can be used to treat pancreatic cancer.

[0016] The present invention also provides the use of the above-mentioned culture method in expanding pancreatic cancer organoids or screening drugs for treating pancreatic cancer.

[0017] The technical solution of the present invention has the following advantages:

[0018] The present invention provides a method for propagating pancreatic cancer organoids, the method comprising a dispersion step; the dispersion step comprises: mechanically dispersing the pancreatic cancer organoids to be propagated by blowing. Studies have found that organoids can achieve almost the same dispersion effect as enzymatic digestion without adding enzyme digestion, relying solely on blowing, and the organoids obtained by mechanical blowing and dispersion have a faster and better growth trend. Therefore, using the propagation method provided by the present invention to propagate organoids has the advantages of short-term rapid growth and economy and high efficiency, can improve the survival rate and growth efficiency of organoids, more quickly reach the number and density of organoids required by the experiment, can shorten the in vitro culture cycle of organoids, improve the culture efficiency of organoids, better accelerate the experimental process, can reduce the waste of consumables such as reagents, and effectively reduce costs.

[0019] Furthermore, the dispersion step includes: blowing and beating the pancreatic cancer organoids to be passaged 10 to 30 times at 0 to 4° C. to mechanically disperse the pancreatic cancer organoids to be passaged. Such conditions are more conducive to the growth of organoids and can prevent the organoids from aging during the passage culture process.

[0020] Furthermore, the dispersion step includes: after discarding the organoid culture medium in the pancreatic cancer organoid culture container, adding Organoid Wash precooled to 0-4°C to the pancreatic cancer organoid culture container to obtain an organoid suspension; after collecting the organoid suspension into a pancreatic cancer organoid dispersion container precooled to 0-4°C, blowing the organoid suspension 10-30 times at 0-4°C to mechanically disperse the pancreatic cancer organoids to be passaged to obtain a dispersed organoid suspension A; after centrifuging the dispersed organoid suspension A at 0-4°C, discarding the supernatant to obtain a pancreatic cancer organoid dispersion container containing organoid precipitate A; adding Organoid Wash precooled to 0-4°C to the pancreatic cancer organoid dispersion container to obtain the organoid suspension; After Wash is added to the pancreatic cancer organoid dispersion container containing organoid precipitate A, the organoid suspension is blown 10 to 30 times at 0 to 4°C to mechanically disperse the pancreatic cancer organoids to be passaged to obtain a dispersed organoid suspension B; after the dispersed organoid suspension B is centrifuged at 0 to 4°C, the supernatant is discarded to obtain a pancreatic cancer organoid dispersion container containing organoid precipitate B. This dispersion step does not require washing of the dispersed organoids, effectively preventing the loss of organoids caused by the washing process of removing the enzyme when using enzyme digestion, and further improving the culture efficiency.

[0021] Further, the passage method also includes a passage step; the passage step is arranged after the dispersion step; the passage step includes: adding matrigel to the pancreatic cancer organoid dispersion container containing organoid precipitate B to obtain a mixture of organoid and matrigel; adding the mixture of organoid and matrigel to the pancreatic cancer organoid culture container, and after the mixture of organoid precipitate and matrigel is condensed and formed, adding the organoid culture medium to the pancreatic cancer organoid culture container to culture the organoid to obtain the pancreatic cancer organoid after passage. The present invention utilizes the temperature-sensitive property of matrigel. When it is below the critical temperature, the matrigel is in a liquid state, which is more conducive to fully mixing with the organoid precipitate. When it is above the critical temperature, the matrigel is in a solid state, which can more effectively simulate the in vivo environment and provide the scaffold required to support cell adhesion and growth. The use of matrigel effectively improves the culture efficiency of pancreatic cancer organoids.

[0022] Furthermore, in the subculturing step, the subculturing ratio of the organoid is 2 to 3: 1. This subculturing ratio is more conducive to the growth of the organoid and can increase the growth rate of the organoid. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Effects of different TrypLE digestion times on the growth trend of organoids under the same conditions (Scale bar = 100 μm).

[0024] Figure 2 : Schematic diagram of the photographic method for counting the number of organoids in the entire dome.

[0025] Figure 3 :Effect of TrypLE digestion time on organoid growth. Figure 3 In the figure, A: The effect of different TrypLE digestion times on the final formation of organoids under the same conditions; B: The statistical results of counting organoids with a diameter greater than or equal to 100 μm.

[0026] Figure 4 :Effect of TrypLE digestion time on organoid viability. Figure 4 In the figure, A: The effect of different TrypLE digestion times on the ATP test results of the same organoid under the same conditions; B: The statistical results of the correlation between TrypLE digestion time and the formation activity of pancreatic cancer organoids.

[0027] Figure 5 :Effect of pipetting times on organoid growth and viability. Figure 5 In the figure, A: The effect of different perfusion times on the growth trend of the same organoid under the same conditions; B: The effect of different perfusion times on the ATP test results of the same organoid under the same conditions.

[0028] Figure 6 : Effects of pipetting solutions on organoid growth and viability. Figure 6 In the figure, A: The effect of different pipetting solutions on the growth trend of organoids under the same conditions; B: The effect of different pipetting solutions on the ATP test results of the same organoid under the same conditions.

[0029] Figure 7 : Effect of passaging ratio on organoid growth and viability. Figure 7 In the figure, A: The effect of different passage ratios on the growth trend of organoids under the same conditions; B: The effect of different passage ratios on the ATP test results of the same organoid under the same conditions. DETAILED DESCRIPTION

[0030] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0031] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0032] Experimental Example 1: Effect of Dispersion Method on Passaging of Pancreatic Cancer Organoids

[0033] This experimental example provides an experiment on the effect of dispersion method on the passage of pancreatic cancer organoids. The experimental process is as follows:

[0034] 1. Culture of Pancreatic Cancer Organoids

[0035] References "Boj, SF, Hwang, CI, Baker, LA, Chio, II, Engle, DD, Corbo, V., Jager, M., Ponz-Sarvise, M., Tiriac, H., Spector, MS, Gracanin, A., Oni, T., Yu, KH, van Boxtel, R., Huch, M., Rivera, KD, Wilson, JP, Feigin, ME, D., Handly-Santana, A., …Tuveson, DA (2015). Organoid models of human and mouse ductal pancreatic cancer. Cell, 160 (1-2), 324-338. "Pancreatic cancer tissue was prepared into pancreatic cancer organoids in a 48-well plate.

[0036] 2. Passaging of Pancreatic Cancer Organoids

[0037] 2.1 Dispersion of Pancreatic Cancer Organoids

[0038] Prepare a 0℃ ice box in advance, pre-cool the Organoid Wash and 1.5mL centrifuge tubes required for subculturing in the ice box, pre-cool the centrifuge to 0℃, and pre-cool the required 96-well plates at 37℃ and 5% (v / v) CO. 2 Pre-warm the sterile cell culture incubator.

[0039] Among them, Organoid Wash is a DMEM basal medium (purchased from Gibco) containing 10mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, purchased from Gibco), 1.0mM glutamine supplement (GlutaMAX Supplement, purchased from Gibco), 100U / mL penicillin-streptomycin (Penicillin-Streptomycin, purchased from Gibco), 100μg / mL primary cell antibiotic Primocin (purchased from InvivoGen), 0.1% (v / v) fetal bovine serum (BSA, purchased from Sigma-Aldrich) and 10μM ROCK-I / II inhibitor Y-27632 (purchased from Selleck).

[0040] 2.1.1 Enzymatic hydrolysis and dispersion under different digestion times

[0041] During passaging, aspirate the organoid culture medium in the 48-well plate along the wall of the well plate, take 600 μL of pre-cooled Organoid Wash and add it to two wells of the 48-well plate at a volume of 300 μL per well, collect the pancreatic cancer organoids in the two wells into pre-cooled 1.5 mL centrifuge tubes to obtain the organoid suspension; after the collection is completed, blow the organoid suspension evenly, divide it into 6 equal parts and put it into 6 1.5 mL centrifuge tubes, with 100 μL in each centrifuge tube;

[0042] The TrypLE digestion time was marked on each centrifuge tube: 0, 3, 6, 9, 12, 15 min. After marking, 200 μL of pre-cooled Oragnorid Wash was added to 6 1.5 mL centrifuge tubes except for the one marked with "0". The remaining centrifuge tubes were blown evenly to obtain a mixture of organoid suspension and Oragnorid Wash or TrypLE. The mixture of organoid suspension and Oragnorid Wash or TrypLE was placed in a 37°C, 5% (v / v) CO 2 Incubate in a sterile cell culture incubator for digestion;

[0043] During the digestion process, take out a centrifuge tube every 3 minutes, and add 1 mL of pre-cooled Oragnorid Wash to the centrifuge tube to terminate the digestion. The centrifuge tube where the digestion is terminated is placed on ice. After the digestion is completed, transfer all centrifuge tubes to a pre-cooled centrifuge and centrifuge at 1400 rpm and 0°C for 5 minutes.

[0044] After centrifugation, remove the supernatant from the centrifuge tube to obtain the centrifuge tube containing the organoid precipitate; place the centrifuge tube containing the organoid precipitate in an ice box, add 1 mL of pre-cooled Oragnorid Wash to each centrifuge tube, then place all the centrifuge tubes in a pre-cooled centrifuge, and centrifuge at 1400 rpm and 0°C for 5 minutes to wash the organoid precipitate;

[0045] The washing operation was repeated once, and after the second washing was completed, the supernatant in the centrifuge tube was aspirated to obtain a centrifuge tube containing an organoid precipitate; the centrifuge tube containing the organoid precipitate was placed in an ice box, and 30 μL of Matrigel matrix gel (purchased from Corning) was added to each centrifuge tube to obtain a mixture of organoid precipitate and matrix gel;

[0046] The mixture of organoid precipitate and matrix gel was added to a preheated 96-well plate at 10 μL per well and placed in a 37°C, 5% (v / v) CO 2 After the mixture of organoid precipitate and matrix gel solidified, 100 μL of organoid culture medium was added to each well of the 96-well plate and the plate was incubated at 37°C and 5% (v / v) CO 2 Cultured in a sterile cell culture incubator.

[0047] During the culture process, the organoid culture medium was replaced every two days, and the growth of pancreatic cancer organoids in the matrix gel was regularly observed and photographed. Figure 1 On the 7th day of culture, the image acquisition, image synthesis and organoid number statistics of the whole dome of the 96-well plate were performed. The photography method used was as follows Figure 2 As shown in the figure, the center is the core, and the field of view of the camera involves four boundaries: top, bottom, left, and right. After the photos are collected, the image analysis software Image J is used to complete the intelligent stitching of the images to form a panoramic image, as shown in the figure. Figure 3 A in the figure, the organoids were counted with a diameter greater than or equal to 100 μm. The results are as follows Figure 3 As shown in B. In addition, using Promega's CellTiter- The 3D Cell Viability Assay measures the viability of pancreatic cancer organoids in 96-well plates by quantifying ATP. Figure 4 A in Figure 1. After ATP quantification, Pearson correlation analysis was performed on the TrypLE digestion time and the viability of pancreatic cancer organoid formation. The results are shown in Figure 1. Figure 4 B in.

[0048] Among them, the ATP quantification process is as follows:

[0049] Place a 96-well plate containing pancreatic cancer organoids and cryopreserved CellTiter- After the 3D reagents were equilibrated to room temperature (25°C), CellTiter- 3D reagent was added to a 96-well plate containing pancreatic cancer organoids at a volume of 100 μL per well to obtain a mixture; the mixture was allowed to stand at room temperature for 5 minutes, and then the mixture in the 96-well plate was transferred in parallel to a white-walled 96-well plate and allowed to stand for another 25 minutes; after the standing period, the fluorescence value was read using an ELISA reader.

[0050] 2.1.2 Mechanical dispersion at different pipetting times

[0051] During passaging, aspirate the organoid culture medium in the 48-well plate along the wall of the well plate, take 600 μL of pre-cooled Organoid Wash and add it to two wells of the 48-well plate at a volume of 300 μL per well, collect the pancreatic cancer organoids in the two wells into pre-cooled 1.5 mL centrifuge tubes to obtain the organoid suspension; after the collection is completed, blow the organoid suspension evenly, divide it into 5 equal portions and place it in 5 1.5 mL centrifuge tubes, with 120 μL in each centrifuge tube;

[0052] Mark the number of times of pipetting on each centrifuge tube: 10, 20, 30, 40, 50 times; after marking, place the centrifuge tube in an ice box and use a pipette to pipette the organoid suspension in the centrifuge tube for different times according to the mark; after pipetting, transfer all centrifuge tubes to a pre-cooled centrifuge and centrifuge at 1400 rpm and 0°C for 5 minutes;

[0053] After centrifugation, remove the supernatant from the centrifuge tube to obtain the centrifuge tube containing the organoid precipitate; place the centrifuge tube containing the organoid precipitate in an ice box, add 1 mL of pre-cooled Oragnorid Wash to each centrifuge tube, and then use a pipette to pipette the organoid suspension in the centrifuge tube for different times according to the label; after pipetting, transfer all centrifuge tubes to a pre-cooled centrifuge and centrifuge at 1400 rpm and 0°C for 5 minutes;

[0054] After the second centrifugation is completed, the supernatant in the centrifuge tube is aspirated to obtain a centrifuge tube containing an organoid precipitate; the centrifuge tube containing the organoid precipitate is placed in an ice box, and 30 μL of Matrigel matrix gel is added to each centrifuge tube to obtain a mixture of organoid precipitate and matrix gel;

[0055] The mixture of organoid precipitate and matrix gel was added to a preheated 96-well plate at 10 μL per well and placed in a 37°C, 5% (v / v) CO 2 After the mixture of organoid precipitate and matrix gel solidified, 100 μL of organoid culture medium was added to each well of the 96-well plate and the plate was incubated at 37°C and 5% (v / v) CO 2 Cultured in a sterile cell culture incubator.

[0056] During the culture process, the organoid culture medium was replaced every two days, and the growth of pancreatic cancer organoids in the matrix gel was regularly observed and photographed. Figure 5 A. On the 7th day of culture, Promega CellTiter- The 3D Cell Viability Assay measures the viability of pancreatic cancer organoids in 96-well plates by quantifying ATP. Figure 5 B in.

[0057] 2.1.3 Mechanical dispersion at different temperatures

[0058] Based on 2.1.2, select 30 times of pipetting and replace the pre-cooled Oragnorid Wash with the Oragnorid Wash at room temperature.

[0059] During the culture process, the organoid culture medium was replaced every two days, and the growth of pancreatic cancer organoids in the matrix gel was regularly observed and photographed. Figure 6 A. On the 7th day of culture, Promega CellTiter- The 3D Cell Viability Assay measures the viability of pancreatic cancer organoids in 96-well plates by quantifying ATP. Figure 6B in.

[0060] 2.1.4 Mechanical dispersion under different pipetting solutions

[0061] Based on 2.1.2, select 30 times of pipetting, and replace the pre-cooled Oragnorid Wash with pre-cooled PBS buffer (concentration 1×, pH 7.4, purchased from Gibco).

[0062] During the culture process, the organoid culture medium was replaced every two days, and the growth of pancreatic cancer organoids in the matrix gel was regularly observed and photographed. Figure 6 A. On the 7th day of culture, Promega CellTiter- 3DCell Viability Assay measures the viability of pancreatic cancer organoids in 96-well plates by quantifying ATP. Figure 6 B in.

[0063] 2.1.5. Passaging at different passaging ratios

[0064] During passaging, aspirate the organoid culture medium in the 48-well plate along the wall of the well plate, take 1000 μL of pre-cooled Organoid Wash and add it to two wells of the 48-well plate at a rate of 500 μL per well, collect the pancreatic cancer organoids in the two wells into pre-cooled 1.5 mL centrifuge tubes to obtain an organoid suspension; after collection, blow and beat the organoid suspension evenly, and take 500 μL, 300 μL, and 200 μL of the organoid suspension into three 1.5 mL centrifuge tubes respectively;

[0065] Mark the subculture ratios on each centrifuge tube: 1:2, 1:3, 1:5 (the centrifuge tube with 500 μL of organoid suspension is marked 1:2, the centrifuge tube with 300 μL of organoid suspension is marked 1:3, and the centrifuge tube with 200 μL of organoid suspension is marked 1:5); after marking, place the centrifuge tube in an ice box and use a pipette to pipette the organoid suspension in the centrifuge tube 30 times; after pipetting, transfer all centrifuge tubes to a pre-cooled centrifuge and centrifuge at 1400 rpm and 0°C for 5 minutes;

[0066] After centrifugation, remove the supernatant from the centrifuge tube to obtain the centrifuge tube containing the organoid precipitate; place the centrifuge tube containing the organoid precipitate in an ice box, add 1 mL of pre-cooled Oragnorid Wash to each centrifuge tube, and then use a pipette to pipette the organoid suspension in the centrifuge tube 30 times according to the label; after pipetting, transfer all centrifuge tubes to a pre-cooled centrifuge and centrifuge at 1400 rpm and 0°C for 5 minutes;

[0067] After the second centrifugation is completed, the supernatant in the centrifuge tube is aspirated to obtain a centrifuge tube containing an organoid precipitate; the centrifuge tube containing the organoid precipitate is placed in an ice box, and 120 μL of Matrigel matrix gel is added to each centrifuge tube to obtain a mixture of organoid precipitate and matrix gel;

[0068] The mixture of organoid precipitate and matrix gel was added to a preheated 96-well plate at 10 μL per well and placed in a 37°C, 5% (v / v) CO 2 After the mixture of organoid precipitate and matrix gel solidified, 100 μL of organoid culture medium was added to each well of the 96-well plate and the plate was incubated at 37°C and 5% (v / v) CO 2 Cultured in a sterile cell culture incubator.

[0069] During the culture process, the organoid culture medium was replaced every two days, and the growth of pancreatic cancer organoids in the matrix gel was observed and photographed regularly. On the 1st, 3rd, 5th, and 7th days of culture, the image acquisition, image synthesis, and organoid number statistics of the entire dome of the 96-well plate were performed. The photography method used was as follows: Figure 2 As shown in the figure, the center is the core, and the field of view of the camera involves four boundaries: top, bottom, left, and right. After the photos are collected, the image analysis software Image J is used to complete the intelligent stitching of the images to form a panoramic image, as shown in the figure. Figure 7 A in the culture. On the 1st, 3rd, 5th and 7th days of culture, Promega CellTiter- The 3D Cell Viability Assay measures the viability of pancreatic cancer organoids in 96-well plates by quantifying ATP. Figure 7 B in.

[0070] 3. Experimental results

[0071] Figure 1 The results showed that pancreatic cancer organoids grew faster in short-term culture without enzyme digestion when the base number was similar, and more organoids with a diameter greater than 100 μm were formed more quickly. Figure 3 It can be clearly seen from the statistics on the right that under the same culture conditions, more pancreatic cancer organoids larger than or equal to 100 μm can be obtained by subculturing without digestive enzyme digestion. Figure 4 It can be clearly seen from the ATP measurement results on the left that without the addition of TrypLE, the growth of organoids has good activity, and there is a certain correlation between the digestion time of TrypLE and the formation activity of pancreatic cancer organoids. Figure 4The Pearson correlation analysis on the right shows that the digestion time of TrypLE digestive enzyme has a certain negative correlation with the formation activity of pancreatic cancer organoids (r=-0.9382, P=0.0056). The longer the digestion time, the worse the activity of the organoids formed in the short term.

[0072] Figure 5 It can be seen that more times of blowing and perfusion can easily cause organoid aging, affecting the final formation and vitality, while 10 to 30 times of blowing and perfusion is more conducive to the growth of organoids and is less prone to aging.

[0073] Figure 6 It can be seen that the use of Organoid Wash in a low temperature environment is more conducive to the maintenance of organoid vitality and growth, while the normal temperature environment and PBS buffer will reduce the vitality of the organoid. On this basis, the experiment in 2.1.4 was repeated at 10 and 20 times of blowing, and the results are shown in Figure 6 B. The conclusions obtained with 10 and 20 times of pipetting were consistent with the conclusion obtained with 30 times of pipetting.

[0074] Figure 7 It can be seen that a subculture ratio of 1:2 or 1:3 is more conducive to the growth of organoids and increases the growth rate, while 1:5 will affect the growth of organoids due to the reduction in initial density.

[0075] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for propagating pancreatic cancer organoids, characterized in that: The passaging method comprises a dispersion step; The dispersion step includes: mechanically dispersing the pancreatic cancer organoids to be passaged by pipetting.

2. The method for subculturing as claimed in claim 1, characterized in that: The dispersing step comprises: blowing and beating the pancreatic cancer organoids to be passaged 10 to 30 times at 0 to 4° C. to mechanically disperse the pancreatic cancer organoids to be passaged.

3. The method for subculturing as claimed in claim 1 or 2, characterized in that: The dispersion step comprises: after discarding the organoid culture medium in the pancreatic cancer organoid culture container, adding Organoid Wash precooled to 0-4°C to the pancreatic cancer organoid culture container to obtain an organoid suspension; after collecting the organoid suspension into a pancreatic cancer organoid dispersion container precooled to 0-4°C, blowing the organoid suspension 10-30 times at 0-4°C to mechanically disperse the pancreatic cancer organoids to be passaged to obtain a dispersed organoid suspension A; after centrifuging the dispersed organoid suspension A at 0-4°C, discarding the supernatant to obtain a pancreatic cancer organoid dispersion container containing the organoid precipitate A; adding Organoid Wash precooled to 0-4°C to the pancreatic cancer organoid dispersion container to obtain the organoid suspension; After Wash is added to the pancreatic cancer organoid dispersion container containing organoid precipitate A, the organoid suspension is blown 10 to 30 times at 0 to 4°C to mechanically disperse the pancreatic cancer organoids to be passaged to obtain a dispersed organoid suspension B; after centrifuging the dispersed organoid suspension B at 0 to 4°C, the supernatant is discarded to obtain a pancreatic cancer organoid dispersion container containing organoid precipitate B.

4. The method for subculturing according to any one of claims 1 to 3, characterized in that: The subculturing method further comprises a subculturing step; The subculturing step is provided after the dispersing step; the subculturing step comprises: adding matrix gel to the pancreatic cancer organoid dispersion container containing the organoid precipitate B to obtain a mixed solution of the organoid and the matrix gel; The mixture of organoids and matrix gel is added to the pancreatic cancer organoid culture container. After the mixture of organoid precipitate and matrix gel is solidified, the organoid culture medium is added to the pancreatic cancer organoid culture container to subculture the organoids to obtain subcultured pancreatic cancer organoids.

5. The method for subculturing as claimed in claim 4, characterized in that: In the passaging step, the passaging ratio of organoids is 2 to 3:

1.

6. The method for subculturing according to any one of claims 3 to 5, characterized in that: The ingredients of the OrganoidWash include N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, glutamine supplement, penicillin-streptomycin, primary cell antibiotic Primocin, fetal bovine serum and ROCK-I / II inhibitor Y-27632.

7. The method for subculturing as claimed in claim 6, characterized in that: The concentration of the N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid in Organoid Wash is 8 to 12 mM; the concentration of the glutamine supplement in Organoid Wash is 1.8 to 2.2 mM; the concentration of penicillin-streptomycin in Organoid Wash is 80 to 120 U / mL; the concentration of the primary cell antibiotic Primocin in Organoid Wash is 80 to 120 μg / mL; in terms of volume percentage, the concentration of the fetal bovine serum in Organoid Wash is 0.08 to 0.12%; the concentration of the ROCK-I / II inhibitor Y-27632 in Organoid Wash is 8 to 12 μM.

8. The method for subculturing according to claim 6 or 7, characterized in that: The components of the OrganoidWash further include a cell culture medium; the cell culture medium includes DMEM basal culture medium, DMEM / F12 basal culture medium and / or AdvancedDMEM / F-12 basal culture medium.

9. A method for screening drugs for treating pancreatic cancer, characterized in that: The method comprises: firstly using the passaging method described in any one of claims 1 to 8 to pass the pancreatic cancer organoids, and then using the pancreatic cancer organoids obtained by passaging as an in vitro model of pancreatic cancer to screen drugs for treating pancreatic cancer.

10. Use of the passaging method according to any one of claims 1 to 8 in expanding pancreatic cancer organoids or screening drugs for treating pancreatic cancer.

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