Polarity reversal stomach organoid buffer solution and construction method and application of polarity reversal stomach organoid buffer solution model

Abstract

The invention provides a polarity reversal stomach organoid buffer solution and a construction method and application of a model of the polarity reversal stomach organoid buffer solution, and belongs to the technical field of organoid culture. The invention provides a polarity reversal buffer solution for stomach organs. The polarity reversal buffer solution comprises the following components: 2mM of EDTA (Ethylene Diamine Tetraacetic Acid), 1mM of KCl, 1% of P / S, 210mu M of Y-27632, 60ng / mL of Wnt-3a, 500ng / mL of R-spondin I and the balance of PBS (Phosphate Buffer Solution). The polarity reversal buffer solution provided by the invention can be well used for polarity reversal of stomach organs, and has the advantages of high reversal success rate, high cell survival rate and short reversal period. According to the polarity reversal stomach organoid model, specific experiment operation can be directly carried out without a microinjection technology, the death rate of the organoid in the operation process is reduced, and it is guaranteed that most polarity reversal organoid is contacted or infected by specific substances in a culture medium.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and relates to organ bionics, and in particular to a polarity-reversed gastric organoid buffer and a construction method and application of a model thereof. Background Art

[0002] The three-dimensional culture of cells requires cells to adapt to their morphology and phenotype, so three-dimensional cell culture is a culture system with good proliferation conditions and functional activity induction. Studies have shown that 3D organoids grown in vitro can reproduce the basic properties of corresponding organs in vivo, becoming a powerful tool for studying injury and repair and regeneration. However, the inward polarity (apical in, AI) of organoids may limit their widespread application in infection models. However, the method of mechanically breaking up organoids to expose the apical surface to the culture medium cannot accurately replicate the infection process and may even cause nonspecific reactions.

[0003] Currently, most techniques used with gastric organoids are microinjection techniques, which require a microinjector, operation learning time, and specific microinjection fluids. During the injection process, the gastric organoids may be punctured or lysed due to operational errors.

[0004] In summary, current gastric organoid culture has the following disadvantages:

[0005] (1) The main disadvantages of existing organoid culture are that it takes a long time for organoids to form, the time from stem cells to formation is long, and it takes a certain amount of time to perform subsequent experimental operations after formation. The construction of polarity-reversed organoids is based on normal organoids, eliminating a series of conventional organoid operations that take longer, so that the polarity surface is reversed to achieve a hierarchical structure similar to human gastric tissue, thereby reducing the operation time.

[0006] (2) The culture conditions are relatively strict, and other operations can only be performed after culturing in expensive matrix gel. In addition, due to the presence of matrix gel, the organoids are wrapped outside and cannot be directly operated. Polarity-reversed organoids only need to be reversed by using a specific reversal buffer, which reduces the difficulty of operation.

[0007] (3) The use of microinjection technology increases the difficulty of operation, and microinjection requires a certain amount of operation time, which can easily lead to the death of organoids. However, since polarity-reversed organoids are suspended in culture medium, they can directly react with corresponding drugs, bacteria, viruses or other accessible substances through the polar surface, thereby achieving the experimental purpose.

[0008] It can be seen that the culture method that reverses the polarity of organoids can more efficiently construct an organoid model. However, for the culture of gastric organoids, the current culture medium makes it difficult to achieve polarity reversal of gastric organoids.

[0009] Some researchers have studied the culture medium for organoid polarity reversal. Although some culture media can reverse the polarity of airway organoids, the survival rate of gastric organoids after polarity reversal is not high. At the same time, it takes a long time to reverse gastric organoids, and gastric organoids after polarity reversal are prone to slow growth, which poses great difficulties for the culture of gastric organoids with reversed polarity.

[0010] Therefore, how to provide a polarity reversal construction method for gastric organoids to overcome the problem that the existing culture medium cannot reverse the polarity of gastric organoids, and even after the polarity reversal, it is easy to cause the survival rate of gastric organoids to be low and the cells after polarity reversal to grow slowly. Summary of the invention

[0011] The present invention is to solve the above technical problems, thereby providing a method for constructing a polarity-reversed gastric organoid model. The technical purpose of the present invention is to solve the problem that the existing culture medium has a low success rate for polarity reversal of gastric organoids, and to solve the problem that after polarity reversal, the survival rate of gastric organoids is low and the cell growth after polarity reversal is slow.

[0012] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0013] The present invention first provides a gastric organoid polarity reversal buffer, which comprises the following components: EDTA 2mM, KCl 1mM, P / S 1%, Y-27632 10μM, Wnt-3a 60 ng / mL, R-spondin I 500ng / mL, and the balance is PBS.

[0014] The present invention also provides a gastric organoid polarity reversal culture medium, which comprises the following components:

[0015]

[0016]

[0017] The present invention further provides a method for constructing a polarity-reversed gastric organoid model, comprising the following steps:

[0018] (1) Selecting gastric organoids that have grown to maturity through primary culture and have a morphological size of 100-150 μm;

[0019] (2) Place the gastric organoids from step (1) into a multi-well culture plate, add 1 mL of pre-cooled PBS to each well to disperse the Dome gel droplets, transfer the suspension to a centrifuge tube, add Cell Recovery Solution to 5 mL, and place in an ice box to incubate for 10 minutes;

[0020] (3) After the incubation is completed, the mixture is taken out and centrifuged at 250×g and 4°C for 5 minutes, the supernatant is removed and the precipitate is retained; then 500 μL of polarity reversal buffer is added, the formula of which is as described in claim 1, the mixture is transferred to an EP tube and incubated on ice for 5 minutes, then centrifuged at 250×g and 4°C for 5 minutes, the supernatant is removed and the precipitate is retained; 500 μL of polarity reversal medium is added to the precipitate, the mixture is resuspended and transferred to a Corning ultra-low adsorption 24-well plate, and the plate is placed on a shaker at 37°C for 5 minutes to allow it to be evenly distributed, then 200 μL of polarity reversal medium is added and the plate is placed in a 37°C incubator for culture; the formula of the polarity reversal medium is as described in claim 2.

[0021] Furthermore, the primary culture step of the gastric organoids in step (1) is:

[0022] Take out the Matrigel matrix gel and melt it. Determine the amount of Matrigel matrix gel to be added according to the amount of precipitated cells, and control the concentration of Dome gel to be not less than 70%; pre-cool the gun tip, use a pipette to resuspend the Matrigel matrix gel and the precipitate to mix them evenly, so that they do not clump or have particles, and minimize the generation of bubbles; then drop the mixed solution in the center of the 24-well culture plate at a volume of 30-50 μL per well, quickly add the mixed solution, and quickly turn it over, and place it in a 37°C incubator for 15 minutes to form stable Dome gel drops; after the Dome gel droplets are formed, take out and add 0.5 mL of complete organoid culture medium containing Y-27632, and add an appropriate amount of PBS to the uninoculated wells around it, and replace the culture medium every 3 days. After one week, replace it with a complete organoid culture medium without Y-27632;

[0023] Wherein, the components of the complete organoid culture medium are:

[0024]

[0025]

[0026] The present invention also provides use of the gastric organoid polarity reversal buffer as described above in constructing a gastric organoid polarity reversal model.

[0027] The present invention also provides use of the gastric organoid polarity reversal culture medium as described above in constructing a gastric organoid polarity reversal model.

[0028] The beneficial effects of the present invention are as follows:

[0029] The polarity reversal gastric organoid model of the present invention can directly perform specific experimental operations without using microinjection technology, reduce the mortality rate of organoids during the operation, and ensure that most polarity reversal organoids are infected with specific substances in the culture medium. The polarity reversal buffer provided by the present invention can be well used for polarity reversal of gastric organoids, and has a high reversal success rate, a high cell survival rate, and a short reversal cycle. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail below in conjunction with the embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still belong to the protection scope of the present invention.

[0031] Example 1

[0032] 1. Formulated as an organoid chelator

[0033] Prepare the chelating agent according to the ingredients and concentrations in Table 1 below and store in a refrigerator at 4°C.

[0034] Table 1 Chelating agent composition and concentration in gastric organoids

[0035]

[0036] 2. Preparation of Gastric Organoid Culture Medium

[0037] Prepare the culture medium according to the ingredients and concentrations in Table 2. Do not freeze-thaw the culture medium repeatedly and use it up within 1 week after preparation.

[0038] Table 2 Composition and concentration of gastric organoid culture medium

[0039]

[0040]

[0041] 3. Primary cell extraction

[0042] The tissue was removed from the cold D Hank's buffer, then rinsed 2-3 times with PBS containing penicillin / streptomycin, and then minced with ophthalmic scissors to a size of 1-2 mm. 3 , collect the tissue into a centrifuge tube using PBS, centrifuge at 250×g for 5 minutes, add 5-10 mL of the prepared chelating agent, mix by inverting upside down, and incubate at 37°C shaker at 220 rpm for 30 minutes.

[0043] After the incubation is completed, the tissue is removed, washed with PBS again for 1-2 times, centrifuged at 250×g for 5 minutes, and then resuspended with 5-10mL PBS. The tissue liquid is filtered through a 100μm filter and the incompletely dissociated tissue is ground with a 1mL syringe head. After repeated flushing with PBS, the filtrate is collected and centrifuged. The supernatant is discarded and the precipitate is retained at the bottom of the centrifuge tube and stored on ice.

[0044] 4. Primary Culture of Gastric Organoids

[0045] Take out the Matrigel matrix gel in advance to melt, and add an appropriate amount of Matrigel matrix gel according to the amount of precipitated cells. In order for the Dome gel droplets to be stably implanted on the bottom of the plate, the concentration should not be less than 70%. Pre-cool the gun tip in advance, use a 100μL pipette to resuspend the Matrigel matrix gel and the precipitate to mix them evenly, so that they do not form clumps or particles, and minimize the generation of bubbles. Then, drop the mixed solution in the center of the 24-well culture plate at a volume of 30-50μL per well, add the mixed solution as quickly as possible, turn it over quickly, and place it in a 37℃ incubator for 15 minutes to form stable Dome gel drops. After the Dome gel droplets are formed, take out and add 0.5mL of complete organoid culture medium (containing Y-27632), and add an appropriate amount of PBS to the uninoculated wells around it. Change the culture medium every 3 days, and replace it with complete culture medium without Y-27632 after one week.

[0046] 5. Construction of gastric polarity-reversed organoids

[0047] Table 3 Components and concentrations of polarity-reversed gastric organoid culture medium

[0048]

[0049]

[0050] Table 4 Reversal buffer composition and concentration

[0051]

[0052] Take out the mature gastric organoids from the incubator and observe whether the organoid morphology is normal under an inverted microscope. The most suitable size is generally around 100-150μm, which has the highest success rate.

[0053] Place in the clean bench and prepare for operation. Use a 1mL pipette to aspirate the culture medium from the bottom edge of the well and discard it. Add 1mL of pre-cooled PBS to each well to disperse the Dome gel droplets. Transfer the suspension to a 15mL centrifuge tube after gently blowing. Then add CellRecovery Solution to the centrifuge tube to 5mL, place it in an ice box, and wait for incubation for 10 minutes.

[0054] Then take it out, centrifuge it at 250×g and 4℃ for 5 minutes, carefully remove the supernatant and keep the precipitate at the bottom. Then add 500μL of polarity reversal buffer and transfer it to a 1.5mL EP tube, incubate it on ice for 5 minutes, then centrifuge it at 250×g and 4℃ for 5 minutes, carefully remove the supernatant and keep the precipitate at the bottom. Add 500μL of complete organoid culture medium to the precipitate, resuspend it and transfer it to Corning ultra-low attachment 24-well plate, place it on a 37℃ shaker for 5 minutes to evenly distribute it, then add 200μL of complete culture medium and place it in a 37℃ incubator for culture.

[0055] Experimental Exploration Example 1

[0056] Exploration method 1: In the present invention, PBS is first used as a buffer to replace the polarity reversal buffer in the present invention. As a result, PBS cannot cause the gastric organoids to reverse their polarity.

[0057] Method 2: The present invention further added 10mM EDTA to the polarity reversal medium. The results showed that only a small number of gastric organoids reversed polarity, and most of them decomposed into single cells. Therefore, the concentration of EDTA was further adjusted to 5mM, 4mM, 2mM, 1mM, and 0.5mM. The results showed that as the concentration decreased, the reversal rate increased, but the efficiency slowed down. At 2mM, the operation could be completed within 5-10 minutes, while at 0.5mM concentration, the number of polarity-reversed organoids was very small.

[0058] On the other hand, although polarity reversal can continue after the single addition of EDTA, the reversal success rate and survival rate are not high, about 40-50%, and it is still easy to decompose into a single cell suspension or appear in the state of cell clumps.

[0059] Exploration method 3: The following exploration process was further conducted on the polarity reversal buffer:

[0060] (1) As organoids are prone to anoikis, two cytokines, Y-27632 and CHIR99021, were added. Y-27632 is a ROCK kinase inhibitor with a good inhibitory effect, while CHIR99021 is a GSK-3α / β inhibitor. Both types of cytokines have a significant effect in inhibiting cell apoptosis.

[0061] When Y-27632 was first added and a concentration gradient distribution was compared from 1-100 μM, it was found that the most suitable concentration for inhibiting apoptosis was 10 μM. At this time, the survival rate of the polarity-reversed organoids was significantly increased to about 70%. At this time, the polarity-reversed organoids could still maintain the polarity-reversed state after 12 hours of culture, while some still underwent apoptosis or disintegrated into single cells.

[0062] After adding CHIR99021, it was found that the probability of organoid anoikis decreased, but the survival rate changed slightly, which was about 50% after counting. It is speculated that CHIR99021 cannot play its full role at this time.

[0063] After the two cytokines were added together, the survival rate did not change much and was still around 70% after counting. It is speculated that the effect of CHIR99021 did not affect the anoikis at this time, but Y-27632 had a greater effect.

[0064] (2) During the organoid culture process, LGR5+ stem cells are closely related to their survival. Three cytokines related to the survival of LGR5+ stem cells, WNT-3a, R-spondin I and Noggin, were further added to the reversal buffer. Among them, WNT-3a and R-spondin I are activating factors of the WNT signaling pathway. After adding them, it was found that the survival rate of the organoids was significantly increased. Their role enabled the stemness of stem cells to be preserved. 24 hours after addition, it was found that the survival rate of the polarity-reversed organoids increased, and the growth trend of the organoids recovered well. Within 48 hours, their size increased significantly. When Noggin was added alone, it did not help the growth of the organoids. During the growth process, the polarity-reversed organoids tended to stick close to each other. Therefore, Noggin was discarded and WNT-3a and R-spondin I were retained.

[0065] (3) Further discovery of K + Ions can accelerate the destruction of tight junctions between cells, causing cell separation, and can also maintain cell activity at a certain concentration. + Identification of concentration. Six commonly used monopotassium, dipotassium or tripotassium compounds, KH2PO4, K2HPO4, K3PO4, KCl, KHCO3, and K2CO3, were selected for comparison. During the study, it was found that high concentrations of phosphate and carbonate easily caused organoids to die during polarity reversal, while low concentrations had no effect, but did not significantly improve the efficiency of polarity reversal. KCl can effectively promote the efficiency of polarity reversal within a certain concentration range, and has a certain positive effect on the survival of organoids within 24 hours. Therefore, we abandoned KH2PO4, K2HPO4, K3PO4, KHCO3, and K2CO3, and chose to add KCl and keep the concentration at 1mM. At this concentration, the efficiency of polarity reversal can be effectively improved, and the time originally required from one to half an hour is reduced to only 10-15 minutes. The choice of KCl reagent can greatly improve the efficiency of polarity reversal operation and reduce the death of organoids caused by operation time.

[0066] (4) During the operation, it was also found that since the organoids were cultured without matrix gel, they were easily affected by the external environment, that is, they were prone to contamination. Therefore, dual antibody P / S was added to prevent contamination.

[0067] (5) When the above reagents were added to the final version of the buffer, we found that the efficiency of polarity reversal was increased to within 5-10 minutes. After the polarity reversal operation, the survival rate of polarity-reversed organoids within 24 hours was significantly improved compared with the initial 5-10%, reaching 90-95%. The growth rate within 48 hours also increased accordingly. After 48 hours, they can be used for in vitro infection experiments or other experiments.

[0068] In summary, the polarity reversal buffer of the present invention has been developed after a large number of experimental explorations, with the addition of corresponding reagents, and through summarizing multiple repeated experiments and failures, the most stable polarity reversal buffer formula with the highest efficiency and success rate for organoid polarity reversal has been obtained.

Claims

1. A gastric organoid polarity reversal buffer, characterized in that The following components were included: EDTA 2 mM, KCl 1 mM, P / S 1%, Y-27632 10 μM, Wnt-3a 60 ng / mL, R-spondin I 500 ng / mL, and the balance was PBS.

2. A gastric organoid polarity reversal culture medium, characterized in that: Includes the following components:

3. A method for constructing a polarity-reversed gastric organoid model, characterized in that: The following steps are involved: (1) Selecting gastric organoids that have grown to maturity through primary culture and have a morphological size of 100-150 μm; (2) Place the gastric organoids from step (1) into a multi-well culture plate, add 1 mL of pre-cooled PBS to each well to disperse the Dome gel droplets, transfer the suspension to a centrifuge tube, add Cell Recovery Solution to 5 mL, and place in an ice box to incubate for 10 minutes; (3) After the incubation is completed, the mixture is taken out and centrifuged at 250×g and 4°C for 5 minutes, the supernatant is removed and the precipitate is retained; then 500 μL of polarity reversal buffer is added, the formula of which is as described in claim 1, the mixture is transferred to an EP tube and incubated on ice for 5 minutes, then centrifuged at 250×g and 4°C for 5 minutes, the supernatant is removed and the precipitate is retained; 500 μL of polarity reversal medium is added to the precipitate, the mixture is resuspended and transferred to a Corning ultra-low adsorption 24-well plate, and the plate is placed on a shaker at 37°C for 5 minutes to allow for even distribution, then 200 μL of polarity reversal medium is added and the plate is placed in an incubator at 37°C for culture; the formula of the polarity reversal medium is as described in claim 2.

4. The method for constructing a polarity-reversed gastric organoid model according to claim 3, characterized in that: The primary culture step of the gastric organoids in step (1) is as follows: Take out the Matrigel matrix gel and melt it. Determine the amount of Matrigel matrix gel to be added according to the amount of precipitated cells, and control the concentration of Dome gel to be not less than 70%; pre-cool the gun tip, use a pipette to resuspend the Matrigel matrix gel and the precipitate to mix them evenly, so that they do not clump or have particles, and minimize the generation of bubbles; then drop the mixed solution in the center of the 24-well culture plate at a volume of 30-50 μL per well, quickly add the mixed solution, and quickly turn it over, and place it in a 37°C incubator for 15 minutes to form stable Dome gel drops; after the Dome gel droplets are formed, take out and add 0.5 mL of complete organoid culture medium containing Y-27632, and add an appropriate amount of PBS to the uninoculated wells around it, and replace the culture medium every 3 days. After one week, replace it with a complete organoid culture medium without Y-27632; Wherein, the components of the complete organoid culture medium are:

5. Use of the gastric organoid polarity reversal buffer according to claim 1 in constructing a gastric organoid polarity reversal model.

6. Use of the gastric organoid polarity reversal culture medium according to claim 2 in the construction of a gastric organoid polarity reversal model.

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