Method for culturing pancreatic cancer and paracancerous organ model

Abstract

The invention relates to a culture method of a pancreatic cancer organ model. The culture method comprises the following steps: cleaning tissues and chopping the tissues through a chopping device; transferring the chopped tissue into a centrifugal tube filled with tissue digestive juice for digestion, and collecting upper suspension and residual tissue precipitate; mixing the collected upper-layer suspension with the digested residual precipitate, and treating to obtain cell and tissue precipitates; embedding the tissues and the cells into matrigel; and after the matrigel is solidified, culturing tissue cells by using a corresponding organoid culture medium, and obtaining primary organoid tissues which are pancreatic cancer tissues. Due to the adoption of the technical scheme, the culture method of the pancreatic cancer organoid model can be used for stably culturing pancreatic cancer organoid with high success rate, and the invention also relates to a culture method of the pancreatic cancer para-carcinoma organoid model.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid model cultivation, and in particular to a method for cultivating a pancreatic cancer and adjacent cancer organoid model. Background Art

[0002] Pancreatic cancer is the 12th most common malignant tumor in the world, with a 5-year survival rate of less than 10%, and most patients die within six months of being diagnosed with pancreatic cancer. Currently, the main treatment for pancreatic cancer is surgery combined with chemotherapy. However, before chemotherapy drugs are used clinically, effective drug sensitivity evaluation methods are urgently needed to quickly predict the effectiveness of treatment plans and adjust treatment strategies in a timely manner to achieve the best clinical efficacy.

[0003] In recent years, organoid technology has been an important technological breakthrough in basic tumor research. Compared with traditional two-dimensional cell culture technology, establishing patient-derived organoid models can more accurately reflect the genetic heterogeneity of tumors, preserve the structure, cellular composition and molecular characteristics of the original tumor, and reproduce the phenotypic characteristics, mutation characteristics and heterogeneity of the original tissue.

[0004] Pancreatic cancer and adjacent normal organoids are considered to be the most difficult types of tumor organoids to culture, so it is very necessary to construct a stable and high-success rate method for culturing pancreatic cancer and adjacent normal organoids. Summary of the invention

[0005] In view of this, an object of the present invention is to provide a method for culturing pancreatic cancer and adjacent cancer organoid models that is stable and has a high success rate.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for culturing a pancreatic cancer organoid model comprises the following steps:

[0008] S1, washing the tissue and chopping the tissue by a chopping device;

[0009] S2, transfer the chopped tissue to a centrifuge tube filled with tissue digestion solution for digestion, and collect the upper suspension and the remaining tissue precipitate;

[0010] S3, mixing the collected upper suspension with the remaining precipitate after digestion and processing to obtain cell and tissue precipitate;

[0011] S4, embedding tissues and cells into matrix gel;

[0012] S5. After the matrix gel solidifies, the tissue cells are cultured using the corresponding organoid culture medium to obtain primary organoids;

[0013] The tissue is pancreatic cancer tissue.

[0014] Furthermore, step S5 further includes a subculture or cryopreservation operation, which is as follows:

[0015] S6. When the density of primary organoids grows to 80%-90%, recover the primary organoids in the matrix gel;

[0016] S7. Resuspend the primary organoids in pre-cooled culture medium and separate the organoids.

[0017] S8. Centrifuge, remove the supernatant, add matrix gel and re-inoculate for subculture; or add organoid freezing solution for storage.

[0018] Furthermore, the tissue cleaning in step S1 includes the following steps:

[0019] S11, placing the obtained tissue into a 10 cm cell culture dish;

[0020] 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.

[0021] Furthermore, the tissue digestion solution in step S2 includes the following components: Advanced DMEM / F-12 culture medium as the basic component, and the following components are added at the final concentration: 10mM HEPEs, 1x GlutaMAX Supplement, 100μg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin, 2.5% FBS, 5mg / ml CollagenaseII, 1mg / ml Dispase II, 10.5μM Y-27632, and 10μg / ml DNAse I.

[0022] Furthermore, in step S2, the chopped tissue is digested by repeated digestion for multiple times;

[0023] For the first digestion: place the tissue fragments in a centrifuge tube containing 3 ml of digestion solution, and digest them in a rotating thermostatic mixer at 37°C, 30 rpm for less than 15 min. Then let it stand for 1 min to collect the upper suspension, and then add 3-4 times the volume of the digestion solution DMEM washing medium to terminate the digestion;

[0024] For each digestion after the first digestion: add 1-2 ml of tissue digestion solution to the tissue sediment left after the last digestion, place it in a rotary thermostatic mixer, digest for 5-10 minutes at 37°C and 30 rpm to obtain a turbid solution, collect the upper suspension after standing for 1 minute, and then add 3-4 times the volume of the digestion solution DMEM washing medium to terminate the digestion;

[0025] Collect any tissue pellet remaining after digestion after the last digestion.

[0026] Further, the organoid culture medium in step S5 includes the following components: Advanced DMEM / F-12 culture medium as the base component, and the following components are added at final concentrations: 50% (V / V) Wnt3a-Conditioned Medium, 10% (V / V) R-spondin1-Conditioned Medium, 10mM HEPEs, 1x GlutaMAX Supplement, 100μg / ml Primocin, 1% (V / V) Penicillin-Streptomycin-Neomycin, 2.5% FBS, 2% (V / V) B-27supplement (50x), 10mM Nicotinamide, 1.25mM N-acetylcysteine, 100ng / mL mNoggin, 50ng / ml mEGF, 100ng / ml hFGF10, 10nM hGastrin I, 500nM A 83-01 and 10.5μM Y-27632.

[0027] Furthermore, the shredding device includes a bracket, a supporting portion, a first mounting plate, a first knife holder and a plurality of first blades, the supporting portion is arranged on the bracket and is used to support tissue, the first mounting plate can be slidably connected to the bracket forward and backward, the first knife holder can be slidably connected to the first mounting plate up and down, the first blade is connected to the first knife holder with the cutting edge facing downward, the length direction of the first blade is along the left-right direction, and the plurality of first blades are arranged at intervals along the front-to-back direction.

[0028] Furthermore, the first blade is a wedge-shaped structure that is larger at the top and smaller at the bottom, and the first blade can be slidably connected to the blade seat front and back, and also includes a first wedge plate and two first elastic parts, the first wedge plates are multiple, and the multiple first wedge plates are arranged in the front and back direction, and the first wedge plates can be slidably connected to the first mounting plate front and back, and there is a first blade between each two adjacent first wedge plates, and the two inclined surfaces of the first blade are respectively matched with the inclined surfaces of the corresponding first wedge plates; one of the first elastic parts is arranged at the front side of the first wedge plate that is most forward, and one of the first elastic parts is used to provide a backward elastic force to the first wedge plate that is most forward, and the other first elastic part is arranged at the rear side of the first wedge plate that is most backward, and the other first elastic part is used to provide a forward elastic force to the first wedge plate that is most backward.

[0029] Furthermore, the support portion includes a second mounting plate, a second knife seat, a plurality of second blades, a plurality of second wedge plates and two second elastic portions, the second mounting plate is arranged on the bracket in a manner that it can slide left and right, the second knife seat can be slidably connected to the second mounting plate up and down, the second blade is a wedge-shaped structure that is small at the top and large at the bottom, the second blade can be slidably connected to the second knife seat left and right with the cutting edge facing upward, the length direction of the second blade is along the front-to-back direction, and the plurality of second blades are arranged at intervals along the left-to-right direction; there are a plurality of second wedge plates, and the plurality of second wedge plates are arranged along the left-to-right direction. The second wedge plates are arranged in a straight line, and the second wedge plates can be slidably connected to the second mounting plate leftward and rightward. A second blade is provided between each two adjacent second wedge plates, and the two inclined surfaces of the second blade are respectively fitted with the inclined surfaces of the corresponding second wedge plates; one of the second elastic parts is arranged on the left side of the second wedge plate that is farthest to the left, and one of the second elastic parts is used to provide a rightward elastic force to the second wedge plate that is farthest to the left, and the other second elastic part is arranged on the right side of the second wedge plate that is farthest to the right, and the other second elastic part is used to provide a leftward elastic force to the wedge plate that is farthest to the right.

[0030] The present invention also provides a method for culturing a pancreatic cancer paracancerous organoid model, comprising the following steps:

[0031] S1, washing the tissue and chopping the tissue by a chopping device;

[0032] S2, transfer the chopped tissue to a centrifuge tube filled with tissue digestion solution for digestion, and collect the upper suspension and the remaining tissue precipitate;

[0033] S3, mixing the collected upper suspension with the remaining precipitate after digestion and processing to obtain cell and tissue precipitate;

[0034] S4, embedding tissues and cells into matrix gel;

[0035] S5. After the matrix gel solidifies, the tissue cells are cultured using the corresponding organoid culture medium to obtain primary organoids;

[0036] The tissue is pancreatic cancer paracancerous tissue.

[0037] The beneficial effects of the present invention are:

[0038] The culture method of the pancreatic cancer organoid model described in the present invention can culture pancreatic cancer organoids stably and with a high success rate, and the culture method of the pancreatic cancer para-cancer organoid model described in the present invention can culture pancreatic cancer para-cancer organoids stably and with a high success rate.

[0039] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a flow chart of the method for culturing a pancreatic cancer organoid model in the present invention;

[0041] Figure 2 This is an example diagram of the growth process of pancreatic cancer organoid P0 in the present invention;

[0042] Figure 3 This is an example of the growth of different wells of pancreatic cancer organoids stably passaged to P3, day 7 in the present invention;

[0043] Figure 4 is a schematic structural diagram of the shredding device of the present invention when viewed from one viewing angle;

[0044] Figure 5 is a schematic structural diagram of the shredding device of the present invention when viewed from another viewing angle;

[0045] Figure 6 This is a flow chart of the method for culturing a pancreatic cancer paracancerous organoid model of the present invention;

[0046] Figure 7 This is an example diagram of the growth process of pancreatic cancer adjacent organoid P0 in the present invention;

[0047] Figure 8 This is the growth status of different wells of the pancreatic cancer adjacent organoids stably passaged to P3 and Day7 in the present invention.

[0048] In the figure: the first mounting plate-1; the first blade-2; the first wedge plate-3; the second mounting plate-4; the second blade-5; the second wedge plate-6; the bottom plate-7; the vertical rod-8; the first limit block-9; the second limit block-10; the first strip hole-11; the third limit block-12; the fourth limit block-13; the second strip hole-14; the first sliding rod-15; the first connecting plate-16; the second sliding rod-17; the second connecting plate-18; the first slide rail-19; the second slide rail-20; the first coil spring-21; the second coil spring-22; the first guide rod-23; the first fixed rod-24; the second guide rod-25; the second fixed rod-26; the third coil spring-27; the fourth coil spring-28; the first waist-shaped hole-29; the second waist-shaped hole-30; the bolt-31; the first vertical plate-32; the second vertical plate-33. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0050] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. 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.

[0051] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0052] 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 usually placed when in use, which is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", etc. are only used to distinguish the description, and cannot be understood as indicating or implying relative importance.

[0053] In addition, the term "same" does not mean that the parts must be absolutely the same, but slight differences are allowed. The term "vertical" only means that the positional relationship between the parts is more vertical than "parallel", and does not mean that the structure must be completely vertical, but can be slightly tilted.

[0054] See also Figure 1 The present invention provides a technical solution: a method for culturing a pancreatic cancer organoid model, comprising the following steps:

[0055] S1, washing the tissue and chopping the tissue by a chopping device;

[0056] The tissue in step S1 is tissue removed by surgery and is pancreatic cancer tissue.

[0057] In this embodiment, the tissue cleaning in step S1 includes the following steps:

[0058] S11, placing the obtained tissue into a 10 cm cell culture dish;

[0059] 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. Impurities such as fat can be removed during the washing process.

[0060] As a preferred embodiment, the mincing in step S1 refers to mincing the tissue into minced meat-like fragments.

[0061] S2, transfer the chopped tissue to a centrifuge tube filled with tissue digestion solution for digestion, and collect the upper suspension and the remaining tissue precipitate;

[0062] The tissue digestion solution in step S2 includes the following components: Advanced DMEM / F-12 culture medium as the basic component, supplemented with the following components at final concentrations: 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.

[0063] The digestion described in step S2 is to place the tissue fragments in a centrifuge tube containing 3 ml of digestion solution and perform digestion treatment in a rotating constant temperature mixer at 37° C. and 30 rpm.

[0064] As a preferred embodiment, the minced tissue is digested in step S2 by repeated digestion;

[0065] For the first digestion: place the tissue fragments in a centrifuge tube containing 3 ml of digestion solution, and digest them in a rotating constant temperature mixer at 37°C, 30 rpm for less than 15 minutes. Then let it stand for 1 minute to collect the upper suspension, and then add DMEM washing medium with 3-4 times the volume of the digestion solution to terminate the digestion.

[0066] For each digestion after the first digestion: add 1-2 ml of tissue digestion solution to the tissue pellet left after the last digestion, place it in a rotary constant temperature mixer, digest at 37°C, 30rpm for 5-10 minutes to obtain a turbid solution, collect the upper suspension after standing for 1 minute, and then add DMEM washing medium 3-4 times the volume of the digestion solution to terminate the digestion.

[0067] Collect any tissue pellet remaining after digestion after the last digestion.

[0068] The chopped tissue is digested multiple times until most of the tissue is digested and only a small amount of tissue fragments remain. The digestion solution can be reduced by half in each digestion after the first digestion. Multiple digestions can avoid overdigestion. Here, overdigestion means that the cells are digested with the digestion solution for too long, resulting in poor cell growth or inability to grow.

[0069] S3, mixing the collected upper suspension with the remaining precipitate after digestion and processing to obtain cell and tissue precipitate;

[0070] As a preferred embodiment, the processing described in step S3 includes the following steps:

[0071] S31. Centrifuge the mixture of the upper suspension and the remaining tissue precipitate after digestion at 4°C, 300g for 5 min, discard the supernatant, and obtain the cell and tissue precipitate.

[0072] S32. Then resuspend the cell and tissue precipitate obtained in step S31 in 10 ml of the above-mentioned washing medium, gently pipette and aspirate several times with a wide-mouth pipette tip, centrifuge at 4°C, 300g for 5 minutes, remove the supernatant, and obtain the cell and tissue precipitate.

[0073] S33. Repeat step S32 once with the cell and tissue precipitate obtained in step S32 to obtain a cell and tissue precipitate.

[0074] S34. Resuspend the cell and tissue precipitate obtained in step S33 in 10 ml of Advanced DMEM / F-12 culture medium, pipette gently several times with a wide-mouth pipette tip, centrifuge at 4°C, 300 g for 5 min, remove the supernatant as much as possible, and obtain the cell and tissue precipitate.

[0075] S4, embedding tissues and cells into matrix gel;

[0076] As a preferred embodiment, the matrix gel embedding described in step S4 can be performed by dropping 20 μl of matrix gel per drop in the center of the wells of the well plate, with 2-3 drops per well.

[0077] As a preferred embodiment, the matrix gel embedding in step S4 is to resuspend the cell / tissue precipitate after centrifugation with Cultrex Reduced Growth Factor Basement Membrane Extract, type 2, add it to a 24-well plate, and incubate the well plate upside down at 37°C to allow the matrix gel embedded with tissue cells to solidify in a dome shape.

[0078] S5. After the matrix gel solidifies, the tissue cells are cultured using the corresponding organoid culture medium to obtain primary organoids;

[0079] The organoid culture medium in step S5 includes the following components: Advanced DMEM / F-12 culture medium as the base component, supplemented with the following components at final concentrations: 50% (V / V) Wnt3a-Conditioned Medium, 10% (V / V) R-spondin1-Conditioned Medium, 10mM HEPEs, 1x GlutaMAX Supplement, 100μg / ml Primocin, 1% (V / V) Penicillin-Streptomycin-Neomycin, 2.5% FBS, 2% (V / V) B-27supplement (50x), 10mM Nicotinamide, 1.25mM N-acetylcysteine, 100ng / mL mNoggin, 50ng / ml mEGF, 100ng / ml hFGF10, 10nM hGastrin I, 500nM A 83-01 and 10.5μM Y-27632.

[0080] As a preferred embodiment, the culture described in step S5 is to place the 24-well plate after the gelatinization in a sterile cell culture incubator at 37° C. and 5% CO 2 for culture.

[0081] As a preferred embodiment, the solidification time of the matrix gel in step S5 depends on the size of the droplets during dispensing, and is usually 30 minutes.

[0082] As a preferred embodiment, step S5 further includes a subculture or cryopreservation operation, which is as follows:

[0083] S6. When the density of primary organoids grows to 80%-90%, remove the original culture medium;

[0084] S7. Resuspend the primary organoids in pre-cooled culture medium and separate the organoids.

[0085] As a preferred embodiment, in the step seven, the organoids are resuspended in cold Advanced DMEM / F-12 culture medium, and the following components are added at final concentrations: 10 mM HEPEs, 1x GlutaMAX Supplement, 100 μg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin, and 2.5% FBS.

[0086] As a preferred embodiment, in step S7, primary organoids are isolated by adding the following components at a final concentration: 10.5 μM Y-27632, 10 μg / ml DNAse I by pipetting with a cold pipette tip or adding TrypLE Express enzyme digestion.

[0087] S8. Centrifuge, remove the supernatant, add matrix gel and re-inoculate for subculture; or add organoid freezing solution for storage.

[0088] The solution involved in this embodiment is prepared as follows:

[0089] Tissue preservation solution was prepared from the following components: DMEM, 1% Penicillin-Streptomycin-Neomycin, and 100 μg / ml Primocin.

[0090] Tissue washing solution was prepared from the following components: PBS, 1% Penicillin-Streptomycin-Neomycin, and 100 μg / ml Primocin.

[0091] Tissue freezing solution is prepared with the following composition: 10% DMSO, 89% FBS, 100 μg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin).

[0092] Tissue digestion solution was prepared from the following components: Advanced DMEM / F-12 medium containing 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.

[0093] DMEM washing medium is prepared with the following components: 1% FBS, 1% Penicillin-Streptomycin-Neomycin, 100 μg / ml Primocin in DMEM medium.

[0094] Ad DMEM / F-12 washing medium was prepared with the following components: Advanced DMEM / F-12 medium containing 10 mM HEPEs, 1x GlutaMAXSupplement, 100 μg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin, and 2.5% FBS.

[0095] Pancreatic cancer organoid culture medium was prepared with the following components: Advanced DMEM / F-12 medium containing 50% (V / V) Wnt3a-Conditioned Medium, 10% (V / V) R-spondin1-Conditioned Medium, 10mM HEPEs, 1x GlutaMAXSupplement, 100μg / ml Primocin, 1% (V / V) Penicillin-Streptomycin-Neomycin, 2.5% FBS, 2% (V / V) B-27supplement (50x), 10mM Nicotinamide, 1.25mM N-acetylcysteine, 100ng / mL mNoggin, 50ng / ml mEGF, 100ng / ml hFGF10, 10nM hGastrin I, 500nM A 83-01, and 10.5μM Y-27632.

[0096] Organoid cryopreservation medium is prepared with the following ingredients: 40% (V / V) Advanced DMEM / F-12 washing medium, 50% (V / V) FBS, and 10% (V / V) DMSO.

[0097] Cultivation experiment 1

[0098] To obtain pancreatic cancer tissue samples from the clinical department of the hospital, take the following steps:

[0099] S1. Take fresh / frozen pancreatic cancer tissue, place it in tissue preservation solution, and transfer it from the operating room to the laboratory biosafety cabinet for operation. Use disposable tweezers to take the tissue out of the preservation solution, place it in tissue cleaning solution, and wash it once. Use a disposable sterile scalpel to cut the tissue into multiple 1-3mm 3 Small pieces. Randomly select a piece of tissue and place it in a cryopreservation tube, add tissue freezing solution, place it in a cryopreservation box at -80℃ for 48 hours, and transfer it to a liquid nitrogen tank for storage. Randomly select a piece of tissue and mince it with a disposable sterile scalpel into a minced meat state.

[0100] S2. Collect pancreatic cancer tissue fragments in a 5ml centrifuge tube, add 3ml tissue digestion solution, and digest in a rotating thermostatic mixer at 37°C, 30rpm. The digestion time is controlled at 15min. After digestion, use a 1ml wide-mouthed gun tip in a biosafety cabinet to repeatedly blow and aspirate to separate the cell clusters from the tissue fragments; then place on wet ice, let stand for 1min, collect the upper suspension into a 15ml centrifuge tube through natural sedimentation, add 3-4 times the volume of digestion solution and cold washing medium, and terminate digestion on ice. If there are more tissue fragments left, digestion can be repeated many times until most of the tissue is digested, leaving only a small amount of tissue fragments, and finally collect the remaining tissue precipitate after digestion.

[0101] S3. The collected upper suspension is mixed with the remaining pellet after digestion and processed to obtain cell and tissue pellets.

[0102] S31. Centrifuge the mixture of the upper suspension and the remaining tissue precipitate after digestion at 4°C, 300g for 5 min, discard the supernatant, and obtain the cell and tissue precipitate.

[0103] S32. Then resuspend the cell and tissue precipitate obtained in step S31 in 10 ml of the above-mentioned washing medium, gently pipette and aspirate several times with a wide-mouth pipette tip, centrifuge at 4°C, 300g for 5 minutes, remove the supernatant, and obtain the cell and tissue precipitate.

[0104] S33. Repeat step S32 once with the cell and tissue precipitate obtained in step S32 to obtain a cell and tissue precipitate.

[0105] S34. Resuspend the cell and tissue precipitate obtained in step S33 in 10 ml of Advanced DMEM / F-12 culture medium, pipette gently several times with a wide-mouth pipette tip, centrifuge at 4°C, 300 g for 5 min, remove the supernatant as much as possible, and obtain the cell and tissue precipitate.

[0106] S4. Resuspend the cell / tissue pellet obtained in step S34 directly with Cultrex Reduced Growth Factor Basement Membrane Extract, type 2, and add them to 24-well plates. Add 20 μl of matrix gel to the center of the wells of the plate, 2-3 drops per well, and incubate upside down at 37°C for 30 min to solidify in a dome shape.

[0107] S5. After the matrix gel solidifies, culture the tissue cells using the corresponding organoid culture medium.

[0108] The cells were cultured in a 24-well plate for 7-10 days, with the medium changed every 2 days. Pancreatic cancer organoid culture medium without Y-27632 was added during the medium change. The growth status of the organoids was observed every day, and photographed and recorded under a microscope to obtain primary pancreatic cancer organoids.

[0109] like Figure 2 As shown, P0 refers to the primary generation. The pancreatic cancer tissue initially isolated from this sample was mainly composed of single cells and smaller cell clusters (A). Obvious organoids could be observed on the third day of growth (B). After continued growth to the sixth day, the diameter of the organoids increased significantly, and some larger organoids could reach a diameter of about 1 mm. Usually, they can reach the level of subculture after 7-10 days of growth.

[0110] S6. Culture for 7-10 days. When the density of primary organoids in the 24-well plate grows to 80%-90%, remove the original culture medium and add cold Advanced DMEM / F-12 culture medium. Use a cold pipette tip to blow and suck to dissociate the matrix gel. Collect the mixture in the well in a 15 ml centrifuge tube and add 2-3 times the volume of cold Advanced DMEM / F-12 culture medium to the centrifuge tube to dilute the mixture of matrix gel and organoids. Place the centrifuge tube in a -20℃ environment for 5 minutes to fully liquefy the matrix gel. Centrifuge at 4℃, 300g for 5 minutes.

[0111] S7. After centrifugation, observe whether the organoids are completely precipitated and whether there is any unremoved matrix gel-primary organoid mixture in the middle layer. If only obvious precipitation is seen, remove the supernatant. Add cold fresh Cultrex Reduced Growth Factor Basement Membrane Extract, type 2 to resuspend the precipitate, re-inoculate at a ratio of 1:2-1:4 and continue culturing; or add organoid freezing solution to the precipitate and freeze it in a liquid nitrogen tank for long-term storage.

[0112] like Figure 3As shown, pancreatic cancer organoids were stably passaged to P3 (i.e., the third generation), and the organoids in different wells grew well on the seventh day.

[0113] See also Figure 4-5 In this embodiment, the shredding device includes a bracket, a supporting portion, a first mounting plate 1, a first knife seat and a plurality of first blades 2. The supporting portion is arranged on the bracket and is used to support the tissue. The first mounting plate 1 can be slidably connected to the bracket forward and backward. The first knife seat can be slidably connected to the first mounting plate 1 up and down. The first blade 2 is connected to the first knife seat with the cutting edge facing downward. The length direction of the first blade 2 is along the left-right direction. The plurality of first blades 2 are arranged at intervals along the front-to-back direction.

[0114] When the tissue needs to be chopped, the tissue to be chopped is placed on the support part, and then the first knife holder is moved up and down on the first mounting plate 1, and the first knife holder drives the first blades 2 to move up and down repeatedly to cut the tissue supported on the support part. In the process of cutting the tissue, the first mounting plate 1 can be slid forward and backward on the support, and the mounting plate drives the first knife holder to move forward and backward, and the first knife holder drives the first blade 2 to move forward and backward. The position of cutting the tissue can be continuously changed, and after a certain period of cutting, the chopped device can chopped the tissue. By cutting the tissue simultaneously with multiple blades, the chopped efficiency of the tissue can be improved.

[0115] In this embodiment, the first blade 2 is a wedge-shaped structure that is larger at the top and smaller at the bottom. The first blade 2 can be slidably connected to the blade seat front and back, and also includes a first wedge plate 3 and two first elastic parts. The first wedge plates 3 are multiple, and the multiple first wedge plates 3 are arranged in the front and back direction. The first wedge plates 3 can be slidably connected to the first mounting plate 1 front and back. There is a first blade 2 between each two adjacent first wedge plates 3, and the two inclined surfaces of the first blade 2 are respectively in contact with the inclined surfaces of the corresponding first wedge plates 3; one of the first elastic parts is arranged at the front side of the first wedge plate 3 that is most forward, and one of the first elastic parts is used to provide a backward elastic force to the first wedge plate 3 that is most forward, and the other first elastic part is arranged at the rear side of the first wedge plate 3 that is most backward, and the other first elastic part is used to provide a forward elastic force to the first wedge plate 3 that is most backward.

[0116] Each of the first blades 2 is sandwiched between two of the first wedge plates 3. During the downward movement of the plurality of first blades 2, each of the first blades 2 can expand the distance between the two first wedge plates 3 that are in contact with it, and the distance between the first wedge plate 3 closest to the front and the wedge plate closest to the rear increases, and the first elastic portion and the second elastic portion are further compressed. After the first blade 2 moves downward for a certain distance, the blade edge of the first blade 2 moves a certain distance away from the lower end of the first wedge plate 3, and the first blade 2 can cut the tissue supported on the support portion.

[0117] During the upward movement of the plurality of first blades 2, under the elastic force of the first elastic part and the second elastic part, the first wedge plate 3 at the front and the first wedge plate 3 at the rear can move toward each other, and each first blade 2 will be squeezed by the two first wedge plates 3 that fit with it. The first wedge plates 3 at both sides of the first blade 2 can keep fit with the first blade 2. After the blade edge of the first blade 2 moves upward to the upper side of the lower end surface of the first wedge plate 3, the lower ends of each two adjacent first wedge plates 3 abut against each other. During the upward movement of the first blade 2, the lower edges of the two first wedge plates 3 that fit with the first blade 2 can scrape the tissues adhered on both sides of the first blade 2, and part of the scraped tissues can fall down again onto the support part. When the first blade 2 moves downward again, the first blade 2 can cut the tissues that fall onto the support part. During the process of cutting tissue using the cutting device, the tissues adhered on both sides of the first blade 2 can be scraped off and cut, and the amount of tissues that are not fully cut can be reduced.

[0118] In this embodiment, the support portion includes a second mounting plate 4, a second knife seat, a plurality of second blades 5, a plurality of second wedge plates 6 and two second elastic portions. The second mounting plate 4 is arranged on the bracket in a manner that it can slide left and right. The second knife seat can be connected to the second mounting plate 4 in a sliding manner up and down. The second blade 5 is a wedge-shaped structure that is small at the top and large at the bottom. The second blade 5 can be connected to the second knife seat in a sliding manner left and right with the cutting edge facing upward. The length direction of the second blade 5 is along the front-to-back direction. The plurality of second blades 5 are arranged at intervals along the left-to-right direction. There are a plurality of second wedge plates 6, and the plurality of second wedge plates 6 are arranged along the left and right directions. The second wedge plates 6 are arranged in the same direction, and the second wedge plates 6 can be slidably connected to the second mounting plate 4 from left to right. There is a second blade 5 between each two adjacent second wedge plates 6, and the two inclined surfaces of the second blade 5 are respectively fitted with the inclined surfaces of the corresponding second wedge plates 6; one of the second elastic parts is arranged on the left side of the second wedge plate 6 that is farthest to the left, and one of the second elastic parts is used to provide a rightward elastic force to the second wedge plate 6 that is farthest to the left, and the other second elastic part is arranged on the right side of the second wedge plate 6 that is farthest to the right, and the other second elastic part is used to provide a leftward elastic force to the wedge plate that is farthest to the right.

[0119] When the first blade 2 cuts the tissue on the supporting portion, the upper end of the second blade 5 is located at the lower side of the upper end surface of the second wedge plate 6. Under the action of the opposing elastic forces provided by the two second elastic portions, the upper ends of the plurality of second wedge plates 6 are pressed against each other, and the upper end surfaces of the plurality of second wedge plates 6 form a first plane, which supports the tissue.

[0120] After the first blade 2 is used to cut tissue for a period of time, the blade edge of the first blade 2 is moved upward to the upper side of the lower end surface of the first wedge plate 3, and the first blade 2 is stopped from moving downward. At this time, the lower ends of each two adjacent first wedge plates 3 abut against each other, and the lower end surfaces of the plurality of first wedge plates 3 form a second plane.

[0121] Then, the second knife holder is moved up and down on the second mounting plate 4, and the second knife holder drives the plurality of second blades 5 to move up and down repeatedly to cut the tissue adhered to the lower end surface of the first wedge-shaped block, that is, to cut the tissue adhered to the second plane. In the process of cutting the tissue adhered to the second plane, the second mounting plate 4 can be slid left and right on the bracket, and the second mounting plate 4 drives the second knife holder to move left and right, and the second knife holder drives the second blades 5 to move left and right. The position of cutting the tissue adhered to the second plane can be continuously changed, and after a certain period of cutting, the shredding device can shred the tissue adhered to the second plane. The amount of tissue that is not fully cut can be further reduced.

[0122] Each of the second blades 5 is sandwiched between two second wedge plates 6. During the upward movement of the plurality of second blades 5, each of the second blades 5 can expand the distance between the two second wedge plates 6 that are in contact with it, increase the distance between the second wedge plate 6 on the leftmost side and the second wedge plate 6 on the rightmost side, and further compress the two second elastic parts. After the second blades 5 move upward for a certain distance, the blade edge of the second blade 5 moves a certain distance away from the upper end of the second wedge plate 6, and the second blade 5 can cut the tissue adhered to the second plane.

[0123] During the downward movement of the plurality of second blades 5, under the elastic force of the two second elastic parts, the second wedge plate 6 on the leftmost side and the second wedge plate 6 on the rightmost side can move toward each other, and each second blade 5 will be squeezed by the two second wedge plates 6 in contact with it, and the second wedge plates 6 on both sides of the second blade 5 can keep in contact with the second blade 5. After the cutting edge of the second blade 5 moves downward to the lower side of the upper end surface of the first wedge plate 6, the upper ends of each two adjacent second wedge plates 6 abut against each other, and the upper end surfaces of the plurality of second wedge plates 6 form the first plane. During the downward movement of the second blade 5, the upper end edges of the two second wedge plates 6 in contact with the second blade 5 can scrape the tissues adhered on both sides of the second blade 5, so as to scrape the tissues adhered on both sides of the second blade 5 to the upper end surface of the second wedge plate 6, that is, the tissues adhered on both sides of the second blade 5 will be scraped onto the first plane.

[0124] The first blade 2 can be cyclically used to cut the tissue on the first plane and the second blade 5 can be used to cut the tissue adhering to the second plane, so as to further reduce the amount of tissue that is not fully cut.

[0125] After the tissue is minced to a desired degree, the minced tissue is scraped off from the first plane and the second plane. At this point, the tissue mincing operation is completed.

[0126] In this embodiment, the bracket includes a bottom plate 7, a vertical rod 8, a first limit block 9 and a second limit block 10. There are two vertical rods 8, and the lower ends of the two vertical rods 8 are fixedly connected to the bottom plate 7. The two vertical rods 8 are fixedly provided with the first limit block 9 and the second limit block 10. The first mounting plate 1 is formed with first strip holes 11 at positions corresponding to the two vertical rods 8. The length direction of the first strip holes 11 is along the front-to-back direction. The two long side edges of the first strip holes 11 are respectively against the two opposite sides of the corresponding vertical rods 8. The lower side of the first mounting plate 1 is against the first limit block 9, and the upper side of the first mounting plate 1 is against the second limit block 10. With this structure, the first mounting plate 1 can be connected to the bracket in a forward and backward sliding manner.

[0127] In this embodiment, the bracket further includes a third limit block 12 and a fourth limit block 13, and the third limit block 12 and the fourth limit block 13 are fixedly provided on the two vertical rods 8. The second mounting plate 4 is formed with a second strip hole 14 at the position corresponding to the two vertical rods 8. The length direction of the second strip hole 14 is along the left-right direction, and the two long side edges of the second strip hole 14 are respectively against the other two opposite sides of the corresponding vertical rod 8. The lower side of the second mounting plate 4 is against the third limit block 12, and the upper side of the second mounting plate 4 is against the fourth limit block 13. With this structure, the second mounting plate 4 can be connected to the bracket in a left-right sliding manner.

[0128] In this embodiment, a first through hole is formed on the first mounting plate 1; the first knife holder includes a first sliding rod 15 slidably fitted in the first through hole and arranged vertically, and a first connecting plate 16 fixedly arranged at the lower end of the first sliding rod 15 and arranged horizontally, and the first blade 2 is connected to the first connecting plate 16. The first knife holder can be moved up and down by simply moving the first sliding rod 15 up and down in the first through hole. With this structure, the first knife holder can be connected to the first mounting plate 1 in a sliding manner up and down.

[0129] In this embodiment, a second through hole is formed on the second mounting plate 4; the second knife holder includes a second sliding rod 17 that is slidably fitted in the second through hole and arranged vertically, and a second connecting plate 18 that is fixed to the upper end of the second sliding rod 17 and arranged horizontally, and the second blade 5 is connected to the second connecting plate 18. The second knife holder can be moved up and down by simply moving the second sliding rod 17 up and down in the second through hole. With this structure, the second knife holder can be connected to the second mounting plate 4 in a sliding manner up and down.

[0130] In this embodiment, the lower side of the first connecting plate 16 is recessed upward to form a first slide groove 19, which is arranged along the front-to-back direction. A plurality of first sliders are slidably fitted in the first slide groove 19, and the first blades 2 are fixedly connected to the plurality of first sliders in a one-to-one correspondence. Specifically, the first blades 2 are connected to the corresponding first sliders through the first vertical plates 32. With this structure, the first blade 2 can be slidably connected to the first blade holder in a front-to-back manner.

[0131] In this embodiment, the upper side surface of the second connecting plate 18 is concave downward to form a second slide groove 20, which is arranged in the left-right direction. A plurality of second sliders are slidably fitted in the second slide groove 20, and the second blades 5 are fixedly connected to the plurality of second sliders in a one-to-one correspondence. Specifically, the second blades 5 are connected to the corresponding second sliders through the second vertical plates 33. With this structure, the second blade 5 can be slidably connected to the second blade holder in a left-right manner.

[0132] In this embodiment, the first knife seat also includes a first coil spring 21 mounted on the first sliding rod 15, the upper end of the first coil spring 21 is fixedly connected to the first mounting plate 1, and the lower end of the first coil spring 21 is fixedly connected to the first connecting plate 16. When the first knife seat is moved downward, the first coil spring 21 is stretched. When the first knife seat needs to be moved upward, the first knife seat can be moved upward under the elastic force of the first coil spring 21, which makes it more convenient to move the first knife seat upward.

[0133] In this embodiment, the second knife seat also includes a second coil spring 22 mounted on the second sliding rod 17, the lower end of the second coil spring 22 is fixedly connected to the second mounting plate 4, and the upper end of the second coil spring 22 is fixedly connected to the second connecting plate 18. When the second knife seat is moved upward, the second coil spring 22 is stretched. When the second knife seat needs to be moved downward, the second knife seat can be moved downward under the elastic force of the second coil spring 22, which makes it more convenient to move the second knife seat downward.

[0134] In this embodiment, two first guide rods 23 are provided on the lower side of the first mounting plate 1. The first guide rods 23 are arranged along the front-to-back direction. The front and rear ends of the first guide rods 23 are fixedly connected to the first mounting plate 1 through first fixing rods 24. First guide holes are formed on the first wedge plate 3 at positions corresponding to the two first guide rods 23. The first guide rods 23 are located in the corresponding first guide holes and match with the first guide holes. With this structure, the first wedge plate 3 can be connected to the first mounting plate 1 in a front-to-back sliding manner.

[0135] In this embodiment, two second guide rods 25 are provided on the upper side of the second mounting plate 4. The second guide rods 25 are arranged in the left-right direction. The left and right ends of the second guide rods 25 are fixedly connected to the second mounting plate 4 through second fixing rods 26. Second guide holes are formed at the positions corresponding to the two second guide rods 25 on the second wedge plate 6. The second guide rods 25 are located in the corresponding second guide holes and matched with the second guide holes. With this structure, the second wedge plate 6 can be connected to the second mounting plate 4 in a left-right sliding manner.

[0136] In this embodiment, one of the first elastic parts includes two third coil springs 27, and the front ends of the two first guide rods 23 are sleeved with the third coil springs 27. The front ends of the third coil springs 27 located at the front ends of the first guide rods 23 abut against the corresponding first fixing rods 24, and the rear ends abut against the first wedge plate 3 located most forward. The other first elastic part also includes two third coil springs 27, and the rear ends of the two first guide rods 23 are sleeved with the third coil springs 27. The rear ends of the third coil springs 27 located at the rear ends of the first guide rods 23 abut against the corresponding first fixing rods 24, and the front ends abut against the first wedge plate 3 located most backward. With this structure, one of the first elastic parts can provide a backward elastic force to the first wedge plate 3 located most forward, and the other first elastic part can provide a forward elastic force to the first wedge plate 3 located most backward.

[0137] In this embodiment, one of the second elastic parts includes two fourth coil springs 28, and the left ends of the two second guide rods 25 are sleeved with the fourth coil spring 28. The left end of the fourth coil spring 28 located at the left end of the second guide rod 25 abuts against the corresponding second fixed rod 26, and the right end abuts against the second wedge plate 6 on the left; the other second elastic part also includes two fourth coil springs 28, and the right ends of the two second guide rods 25 are sleeved with the fourth coil spring 28. The right end of the fourth coil spring 28 located at the right end of the second guide rod 25 abuts against the corresponding second fixed rod 26, and the left end abuts against the second wedge plate 6 on the right. With this structure, one of the second elastic parts can provide a rightward elastic force to the second wedge plate 6 on the left, and the other second elastic part can provide a leftward elastic force to the first wedge plate 3 on the right.

[0138] In this embodiment, a first waist-shaped hole 29 is formed at a position of the first vertical plate 32 corresponding to the first guide rod 23, and the length direction of the first waist-shaped hole 29 is along the vertical direction, and the first guide rod 23 is located in the first waist-shaped hole 29. With this structure, the first guide rod 23 will not hinder the first vertical plate 32 and the first blade 2 from moving up and down.

[0139] In this embodiment, a second waist-shaped hole 30 matching with the second guide rod 25 is formed at a position of the second vertical plate 33 corresponding to the second guide rod 25, the length direction of the second waist-shaped hole 30 is along the vertical direction, and the second guide rod 25 is located in the second waist-shaped hole 30. With this structure, the second guide rod 25 will not hinder the second vertical plate 33 and the second blade 5 from moving up and down.

[0140] In this embodiment, a screw hole is formed on the vertical rod 8 at a position corresponding to the second limit block 10, and a through hole is formed on the second limit block 10 at a position corresponding to the screw hole. A bolt 31 passes through the through hole and is screwed into the screw hole, and the bolt head of the bolt 31 faces the vertical rod 8 to abut against the second limit block 10. The bolt 31 is unscrewed from the screw hole, and the second limit block 10 is removed from the vertical rod 8. At this time, the first mounting plate 1 and other structural components connected to the first mounting plate 1 can be removed from the bracket. When it is necessary to place tissue on the first plane, and when it is necessary to scrape off tissue fragments on the first plane and the second plane, the first mounting plate 1 and other structural components connected to the first mounting plate 1 can be removed from the bracket, which is more convenient when placing tissue on the first plane and scraping off tissue fragments on the first plane and the second plane.

[0141] See also Figure 6 The present invention also provides a technical solution: a method for culturing a pancreatic cancer paracancerous organoid model, comprising the following steps:

[0142] S1, washing the tissue and chopping the tissue by a chopping device;

[0143] The tissue in step S1 is tissue removed by surgery. The tissue is pancreatic cancer adjacent tissue.

[0144] In this embodiment, the tissue cleaning in step S1 includes the following steps:

[0145] S11, placing the obtained tissue into a 10 cm cell culture dish;

[0146] 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. Impurities such as fat can be removed during the washing process.

[0147] As a preferred embodiment, the mincing in step S1 refers to mincing the tissue into minced meat-like fragments.

[0148] S2, transfer the chopped tissue to a centrifuge tube filled with tissue digestion solution for digestion, and collect the upper suspension and the remaining tissue precipitate;

[0149] The tissue digestion solution in step S2 includes the following components: Advanced DMEM / F-12 culture medium as the basic component, supplemented with the following components at final concentrations: 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.

[0150] The digestion described in step S2 is to place the tissue fragments in a centrifuge tube containing 3 ml of digestion solution and perform digestion treatment in a rotating constant temperature mixer at 37° C. and 30 rpm.

[0151] As a preferred embodiment, the minced tissue is digested in step S2 by repeated digestion;

[0152] For the first digestion: place the tissue fragments in a centrifuge tube containing 3 ml of digestion solution, and digest them in a rotating constant temperature mixer at 37°C, 30 rpm for less than 15 minutes. Then let it stand for 1 minute to collect the upper suspension, and then add DMEM washing medium with 3-4 times the volume of the digestion solution to terminate the digestion.

[0153] For each digestion after the first digestion: add 1-2 ml of tissue digestion solution to the tissue pellet left after the last digestion, place it in a rotary constant temperature mixer, digest at 37°C, 30rpm for 5-10 minutes to obtain a turbid solution, collect the upper suspension after standing for 1 minute, and then add DMEM washing medium 3-4 times the volume of the digestion solution to terminate the digestion.

[0154] Collect any tissue pellet remaining after digestion after the last digestion.

[0155] After the chopped tissue is digested for multiple times, it is best to digest most of the tissue and leave only a small amount of tissue fragments. The digestion solution can be reduced by half in each digestion after the first digestion. Multiple digestions can be performed. S3. The collected upper suspension is mixed with the remaining precipitate after digestion and processed to obtain cell and tissue precipitates;

[0156] As a preferred embodiment, the processing described in step S3 includes the following steps:

[0157] S31. Centrifuge the mixture of the upper suspension and the remaining tissue precipitate after digestion at 4°C, 300g for 5 min, discard the supernatant, and obtain the cell and tissue precipitate.

[0158] S32. Then resuspend the cell and tissue precipitate obtained in step S31 in 10 ml of the above-mentioned washing medium, gently pipette and aspirate several times with a wide-mouth pipette tip, centrifuge at 4°C, 300g for 5 minutes, remove the supernatant, and obtain the cell and tissue precipitate.

[0159] S33. Repeat step S32 once with the cell and tissue precipitate obtained in step S32 to obtain a cell and tissue precipitate.

[0160] S34. Resuspend the cell and tissue precipitate obtained in step S33 in 10 ml of Advanced DMEM / F-12 culture medium, pipette gently several times with a wide-mouth pipette tip, centrifuge at 4°C, 300 g for 5 min, remove the supernatant as much as possible, and obtain the cell and tissue precipitate.

[0161] S4, embedding tissues and cells into matrix gel;

[0162] As a preferred embodiment, the matrix gel embedding described in step S4 can be performed by dropping 20 μl of matrix gel per drop in the center of the wells of the well plate, with 2-3 drops per well.

[0163] As a preferred embodiment, the matrix gel embedding in step S4 is to resuspend the cell / tissue precipitate after centrifugation with Cultrex Reduced Growth Factor Basement Membrane Extract, type 2, add it to a 24-well plate, and incubate the well plate upside down at 37°C to allow the matrix gel embedded with tissue cells to solidify in a dome shape.

[0164] S5. After the matrix gel solidifies, the tissue cells are cultured using the corresponding organoid culture medium to obtain primary organoids;

[0165] The organoid culture medium in step S5 includes the following components: Advanced DMEM / F-12 culture medium as the base component, and the following components are added at the final concentration: 50% (V / V) Wnt3a-Conditioned Medium, 10% (V / V) R-spondin1-Conditioned Medium, 10mM HEPEs, 1x GlutaMAX Supplement, 100μg / ml Primocin, 1% (V / V) Penicillin-Streptomycin-Neomycin, 2.5% FBS, 2% (V / V) B-27supplement (50x), 10mM Nicotinamide, 1.25mM N-acetylcysteine, 100ng / mL mNoggin, 50ng / ml mEGF, 100ng / ml hFGF10, 10nM hGastrin I, 500nM A 83-01, 10.5μM Y-27632 and 1 μM PGE2.

[0166] As a preferred embodiment, the culture described in step S5 is to place the 24-well plate after the gelatinization in a sterile cell culture incubator at 37° C. and 5% CO 2 for culture.

[0167] As a preferred embodiment, the solidification time of the matrix gel in step S5 depends on the size of the droplets during dispensing, and is usually 30 minutes.

[0168] As a preferred embodiment, step S5 further includes a subculture or cryopreservation operation, which is as follows:

[0169] S6. When the density of primary organoids grows to 80%-90%, remove the original culture medium;

[0170] S7. Resuspend the primary organoids in pre-cooled culture medium and separate the organoids.

[0171] As a preferred embodiment, in step S7, the organoids are resuspended in cold Advanced DMEM / F-12 culture medium, and the following components are added at final concentrations: 10 mM HEPEs, 1x GlutaMAX Supplement, 100 μg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin, and 2.5% FBS.

[0172] As a preferred embodiment, in step seven, primary organoids are isolated by adding the following components at final concentrations: 10.5 μM Y-27632, 10 μg / ml DNAse I, by pipetting with a cold pipette tip or adding TrypLE Express enzyme digestion.

[0173] S8. Centrifuge, remove the supernatant, add matrix gel and re-inoculate for subculture; or add organoid freezing solution for storage.

[0174] The solution involved in this embodiment is prepared as follows:

[0175] Tissue preservation solution was prepared from the following components: DMEM, 1% Penicillin-Streptomycin-Neomycin, and 100 μg / ml Primocin.

[0176] Tissue washing solution was prepared from the following components: PBS, 1% Penicillin-Streptomycin-Neomycin, and 100 μg / ml Primocin.

[0177] Tissue freezing solution is prepared with the following composition: 10% DMSO, 89% FBS, 100 μg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin).

[0178] Tissue digestion solution was prepared from the following components: Advanced DMEM / F-12 medium containing 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.

[0179] DMEM washing medium is prepared with the following components: 1% FBS, 1% Penicillin-Streptomycin-Neomycin, 100 μg / ml Primocin in DMEM medium.

[0180] Ad DMEM / F-12 washing medium was prepared with the following components: Advanced DMEM / F-12 medium containing 10 mM HEPEs, 1x GlutaMAXSupplement, 100 μg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin, and 2.5% FBS.

[0181] Pancreatic cancer paracancerous organoid culture medium was prepared with the following components: 50% (V / V) Wnt3a-Conditioned Medium, 10% (V / V) R-spondin1-Conditioned Medium, 10mM HEPEs, 1x GlutaMAX Supplement, 100μg / ml Primocin, 1% (V / V) Penicillin-Streptomycin-Neomycin, 2.5% FBS, 2% (V / V) B-27 supplement (50x), 10mM Nicotinamide, 1.25mM N-acetylcysteine, 100ng / mL mNoggin, 50ng / ml mEGF, 100ng / ml hFGF10, 10nM hGastrin I, 500nM A 83-01, 10.5μM Y-27632, 1μM PGE2 Advanced DMEM / F-12 medium.

[0182] Organoid cryopreservation medium is prepared with the following ingredients: 40% (V / V) Advanced DMEM / F-12 washing medium, 50% (V / V) FBS, and 10% (V / V) DMSO.

[0183] Cultivation experiment 1

[0184] Obtain pancreatic cancer adjacent tissue samples from the hospital clinical department and take the following steps:

[0185] S1. Take fresh / frozen pancreatic cancer adjacent tissue, place it in tissue preservation solution, and transfer it from the operating room to the laboratory biosafety cabinet for operation. Use disposable tweezers to take the tissue out of the preservation solution, place it in tissue cleaning solution, and wash it once. Use a disposable sterile scalpel to cut the tissue into multiple 1-3mm 3 Small pieces. Randomly select a piece of tissue and place it in a cryopreservation tube, add tissue freezing solution, place it in a cryopreservation box at -80℃ for 48 hours, and transfer it to a liquid nitrogen tank for storage. Randomly select a piece of tissue and mince it with a disposable sterile scalpel into a minced meat state.

[0186] S2. Collect pancreatic cancer adjacent tissue fragments in a 5ml centrifuge tube, add 3ml tissue digestion solution, and digest in a rotating thermostatic mixer at 37°C, 30rpm. The digestion time is controlled at 15min. After digestion, use a 1ml wide-mouthed gun tip in a biosafety cabinet to repeatedly blow and aspirate to separate the cell clusters from the tissue fragments; then place on wet ice, let stand for 1min, collect the upper suspension into a 15ml centrifuge tube through natural sedimentation, add 3-4 times the volume of digestion solution and cold washing medium, and terminate digestion on ice. If there are more tissue fragments left, digestion can be repeated several times until most of the tissue is digested, leaving only a small amount of tissue fragments, and finally collect the remaining tissue precipitate after digestion.

[0187] S3. The collected upper suspension is mixed with the remaining pellet after digestion and processed to obtain cell and tissue pellets.

[0188] S31. Centrifuge the mixture of the upper suspension and the remaining tissue precipitate after digestion at 4°C, 300g for 5 min, discard the supernatant, and obtain the cell and tissue precipitate.

[0189] S32. Then resuspend the cell and tissue precipitate obtained in step S31 in 10 ml of the above-mentioned washing medium, gently pipette and aspirate several times with a wide-mouth pipette tip, centrifuge at 4°C, 300g for 5 minutes, remove the supernatant, and obtain the cell and tissue precipitate.

[0190] S33. Repeat step S32 once with the cell and tissue precipitate obtained in step S32 to obtain a cell and tissue precipitate.

[0191] S34. Resuspend the cell and tissue precipitate obtained in step S33 in 10 ml of Advanced DMEM / F-12 culture medium, pipette gently several times with a wide-mouth pipette tip, centrifuge at 4°C, 300 g for 5 min, remove the supernatant as much as possible, and obtain the cell and tissue precipitate.

[0192] S4. Resuspend the cell / tissue pellet obtained in step S34 directly with Cultrex Reduced Growth Factor Basement Membrane Extract, type 2, and add them to 24-well plates. Add 20 μl of matrix gel to the center of the wells of the plate, 2-3 drops per well, and incubate upside down at 37°C for 30 min to solidify in a dome shape.

[0193] S5. After the matrix gel solidifies, culture the tissue cells using the corresponding organoid culture medium.

[0194] The cells were cultured in a 24-well plate for 7-10 days, with the medium changed every 2 days. Pancreatic cancer para-cancer organoid culture medium without Y-27632 was added during the medium change. The growth status of the organoids was observed every day, and photographed and recorded under a microscope to obtain primary pancreatic cancer para-cancer organoids.

[0195] like Figure 7 As shown, P0 refers to the primary generation. The pancreatic cancer adjacent tissue initially isolated from this sample was mainly composed of single cells and smaller cell clusters (A). Obvious organoids could be observed on the third day of growth (B). After continued growth to the sixth day, the diameter of the organoids increased significantly, and some larger organoids could reach a diameter of about 1 mm. Usually, they can reach the level of subculture after 7-10 days of growth.

[0196] S6. Culture for 7-10 days. When the density of primary organoids in the 24-well plate grows to 80%-90%, remove the original culture medium and add cold Advanced DMEM / F-12 culture medium. Use a cold pipette tip to blow and suck to dissociate the matrix gel. Collect the mixture in the well in a 15 ml centrifuge tube and add 2-3 times the volume of cold Advanced DMEM / F-12 culture medium to the centrifuge tube to dilute the mixture of matrix gel and organoids. Place the centrifuge tube in a -20℃ environment for 5 minutes to fully liquefy the matrix gel. Centrifuge at 4℃, 300g for 5 minutes.

[0197] S7. After centrifugation, observe whether the organoids are completely precipitated and whether there is any unremoved matrix gel-primary organoid mixture in the middle layer. If only obvious precipitation is seen, remove the supernatant. Add cold fresh Cultrex Reduced Growth Factor Basement Membrane Extract, type 2 to resuspend the precipitate, re-inoculate at a ratio of 1:2-1:4 and continue culturing; or add organoid freezing solution to the precipitate and freeze it in a liquid nitrogen tank for long-term storage.

[0198] like Figure 8 As shown, pancreatic cancer paracancerous organoids were stably passaged to P3 (the third generation), and the organoids in different wells grew well on the seventh day.

[0199] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution 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 solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for culturing a pancreatic cancer organoid model, characterized in that: The following steps are involved: S1, washing the tissue and chopping the tissue by a chopping device; S2, transfer the chopped tissue to a centrifuge tube filled with tissue digestion solution for digestion, and collect the upper suspension and the remaining tissue precipitate; S3, mixing the collected upper suspension with the remaining precipitate after digestion and processing to obtain cell and tissue precipitate; S4, embedding tissues and cells into matrix gel; S5. After the matrix gel solidifies, the tissue cells are cultured using the corresponding organoid culture medium to obtain primary organoids; The tissue is pancreatic cancer tissue.

2. The method for culturing a pancreatic cancer organoid model according to claim 1, characterized in that: Step S5 also includes subculture or cryopreservation operations, which are as follows: S6. When the density of primary organoids grows to 80%-90%, recover the primary organoids in the matrix gel; S7. Resuspend the primary organoids in pre-cooled culture medium and separate the organoids. S8. Centrifuge, remove the supernatant, add matrix gel and re-inoculate for subculture; or add organoid freezing solution for storage.

3. The method for culturing a pancreatic cancer organoid model according to claim 1, characterized in that: The tissue cleaning in step S1 comprises the following steps: S11, placing the obtained tissue into a 10 cm 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 a pancreatic cancer organoid model according to claim 1, characterized in that: The tissue digestion solution in step S2 includes the following components: Advanced DMEM / F-12 culture medium as the basic component, and the following components are added at the final concentration: 10mM HEPEs, 1x GlutaMAX Supplement, 100μg / ml Primocin, 1% Penicillin-Streptomycin-Neomycin, 2.5% FBS, 5mg / ml Collagenase II, 1mg / ml DispaseII, 10.5μMY-27632, 10μg / ml DNAse I.

5. The method for culturing a pancreatic cancer organoid model according to claim 1, characterized in that: In step S2, the chopped tissue is digested by repeated digestion multiple times; For the first digestion: place the tissue fragments in a centrifuge tube containing 3 ml of digestion solution, and digest them in a rotating thermostatic mixer at 37°C, 30 rpm for less than 15 min. Then let it stand for 1 min to collect the upper suspension, and then add 3-4 times the volume of the digestion solution DMEM washing medium to terminate the digestion; For each digestion after the first digestion: add 1-2 ml of tissue digestion solution to the tissue sediment left after the last digestion, place it in a rotary thermostatic mixer, digest for 5-10 minutes at 37°C and 30 rpm to obtain a turbid solution, collect the upper suspension after standing for 1 minute, and then add 3-4 times the volume of the digestion solution DMEM washing medium to terminate the digestion; Collect any tissue pellet remaining after digestion after the last digestion.

6. The method for culturing a pancreatic cancer organoid model according to claim 1, characterized in that: The organoid culture medium in step S5 includes the following components: Advanced DMEM / F-12 culture medium as the base component, supplemented with the following components at final concentrations: 50% (V / V) Wnt3a-Conditioned Medium, 10% (V / V) R-spondin1-Conditioned Medium, 10mM HEPEs, 1x GlutaMAX Supplement, 100μg / ml Primocin, 1% (V / V) Penicillin-Streptomycin-Neomycin, 2.5% FBS, 2% (V / V) B-27supplement (50x), 10mM Nicotinamide, 1.25mM N-acetylcysteine, 100ng / mL mNoggin, 50ng / ml mEGF, 100ng / ml hFGF10, 10nM hGastrin I, 500nM A 83-01 and 10.5μM Y-27632.

7. The method for culturing a pancreatic cancer organoid model according to claim 1, characterized in that: The shredding device includes a bracket, a supporting portion, a first mounting plate, a first knife holder and a plurality of first blades. The supporting portion is arranged on the bracket and is used to support tissue. The first mounting plate can be slidably connected to the bracket forward and backward. The first knife holder can be slidably connected to the first mounting plate upward and downward. The first blade is connected to the first knife holder with the cutting edge facing downward. The length direction of the first blade is along the left-right direction. The plurality of first blades are arranged at intervals along the front-back direction.

8. The method for culturing a pancreatic cancer organoid model according to claim 1, characterized in that: The first blade is a wedge-shaped structure that is larger at the top and smaller at the bottom. The first blade can be slidably connected to the blade seat front and back, and also includes a first wedge plate and two first elastic parts. The first wedge plates are multiple, and the multiple first wedge plates are arranged in the front and back direction. The first wedge plates can be slidably connected to the first mounting plate front and back. There is a first blade between each two adjacent first wedge plates, and the two inclined surfaces of the first blade are respectively in contact with the inclined surfaces of the corresponding first wedge plates; one of the first elastic parts is arranged at the front side of the first wedge plate that is most forward, and one of the first elastic parts is used to provide a backward elastic force to the first wedge plate that is most forward, and the other first elastic part is arranged at the rear side of the first wedge plate that is most backward, and the other first elastic part is used to provide a forward elastic force to the first wedge plate that is most backward.

9. The method for culturing a pancreatic cancer organoid model according to claim 1, characterized in that: The support portion includes a second mounting plate, a second knife seat, a plurality of second blades, a plurality of second wedge plates and two second elastic portions, the second mounting plate is arranged on the bracket in a manner that it can slide left and right, the second knife seat can be slidably connected to the second mounting plate up and down, the second blade is a wedge-shaped structure that is small at the top and large at the bottom, the second blade can be slidably connected to the second knife seat left and right with the cutting edge facing upward, the length direction of the second blade is along the front-to-back direction, and the plurality of second blades are arranged at intervals along the left-to-right direction; there are a plurality of second wedge plates, and the plurality of second wedge plates are arranged along the left-to-right direction The second wedge plate can be slidably connected to the second mounting plate leftward and rightward, and a second blade is provided between each two adjacent second wedge plates, and the two inclined surfaces of the second blade are respectively fitted with the inclined surfaces of the corresponding second wedge plates; one of the second elastic parts is arranged on the left side of the second wedge plate farthest to the left, and one of the second elastic parts is used to provide a rightward elastic force to the second wedge plate farthest to the left, and the other second elastic part is arranged on the right side of the second wedge plate farthest to the right, and the other second elastic part is used to provide a leftward elastic force to the wedge plate farthest to the right.

10. A method for culturing a pancreatic cancer paracancerous organoid model, characterized in that: The following steps are involved: S1, washing the tissue and chopping the tissue by a chopping device; S2, transfer the chopped tissue to a centrifuge tube filled with tissue digestion solution for digestion, and collect the upper suspension and the remaining tissue precipitate; S3, mixing the collected upper suspension with the remaining precipitate after digestion and processing to obtain cell and tissue precipitate; S4, embedding tissues and cells into matrix gel; S5. After the matrix gel solidifies, the tissue cells are cultured using the corresponding organoid culture medium to obtain primary organoids; The tissue is pancreatic cancer paracancerous tissue.

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