A high-efficiency organ-like pollution inhibition reagent

Abstract

This invention belongs to the field of organoid culture technology, specifically relating to a highly efficient organoid anticontamination reagent. This highly efficient organoid anticontamination reagent is composed of Primocin, amphotericin B, and Y-27632. In the organoid culture medium system, the final concentration of Primocin is 20-70 μg / mL, the final concentration of amphotericin B is 0.1-1 μg / mL, and the final concentration of Y-27632 is 5-20 μM. The highly efficient organoid anticontamination reagent provided by this invention exhibits dual high-efficiency inhibition against both bacteria and fungi, has a broad anticontamination spectrum, and is not prone to inducing microbial resistance. Simultaneously, it can reduce cytotoxicity, and long-term use does not alter the expression of organoid-specific markers, thus maintaining differentiation function.

Description

Technical Field

[0001] This invention belongs to the field of organoid culture technology, specifically relating to a highly efficient organoid anticontamination reagent. Background Technology

[0002] Organoids, as three-dimensional cultured cell structures, can mimic the cellular composition and functional characteristics of real organs and are widely used in disease model construction, drug screening, and regenerative medicine research. Organoid culture requires extremely sterile environments, and microbial contamination is one of the main causes of culture failure.

[0003] The existing methods for inhibiting contamination commonly used in organoid culture have the following main defects: (1) The spectrum of contamination inhibition is narrow. A single antibiotic or antifungal agent can only inhibit one of the bacteria or fungi, making it difficult to deal with mixed contamination; (2) Long-term use of a single agent can easily induce microbial resistance and reduce the effect of contamination inhibition; (3) Some contamination inhibitors are cytotoxic to organoid stem cells at effective concentrations, inhibiting adhesion, proliferation, and even inducing apoptosis; (4) Existing contamination inhibitors have poor compatibility with organoid composite culture media and are prone to interact with the components of the culture media, affecting the effect of contamination inhibition or damaging the nutrient system; (5) The operation of composite contamination inhibitor formulations is complicated, increasing the risk of contamination. Summary of the Invention

[0004] To address the problems of narrow anticontamination spectrum, easy induction of drug resistance, high cytotoxicity, and poor compatibility in existing technologies, this invention provides a highly efficient organoid anticontamination reagent that achieves broad anticontamination spectrum, no cytotoxicity, strong compatibility, and simple operation, which is of great significance for improving the success rate of organoid in vitro culture.

[0005] The technical problem solved by this invention is achieved by the following technical solution:

[0006] The purpose of this invention is to provide a highly efficient organoid anticontamination reagent, which is composed of Primocin, amphotericin B and Y-27632.

[0007] Furthermore, in the organoid culture system, the final concentration of Primocin was 20-70 μg / mL, the final concentration of amphotericin B was 0.1-1 μg / mL, and the final concentration of Y-27632 was 5-20 μM.

[0008] Furthermore, in the organoid culture medium system, the final concentration of Primocin was 50 μg / mL, the final concentration of amphotericin B was 0.25 μg / mL, and the final concentration of Y-27632 was 10 μM.

[0009] Furthermore, the anti-pollution reagent is added in the form of a mother liquor, wherein:

[0010] Primocin stock solution was prepared using sterile PBS buffer, with a stock solution concentration of 50 mg / mL;

[0011] The stock solution of amphotericin B was prepared using sterile DMSO, with a concentration of 0.25 mg / mL.

[0012] Y-27632 stock solution was prepared using sterile DMSO with a concentration of 100 mM.

[0013] Furthermore, organoids include intestinal organoids, liver organoids, kidney organoids, lung organoids, intrahepatic bile duct carcinoma organoids, hepatocellular carcinoma organoids, and lung cancer organoids.

[0014] Furthermore, the anti-contamination reagent is added to the organoid culture medium system either as a single solution or as a mixed solution.

[0015] Contamination inhibitors are used in the primary, passaged, or long-term culture of organoids, covering the entire organoid culture process. They can significantly improve the success rate of in vitro organoid culture.

[0016] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0017] 1. The highly efficient organoid anticontamination reagent provided by this invention achieves dual high-efficiency inhibition of bacteria and fungi through the synergistic effect of the three components: Primocin, amphotericin B, and Y-27632. It has a broad spectrum of anticontamination and is not prone to inducing microbial resistance. The addition of Y-27632 not only helps to enhance the anticontamination effect, but also effectively inhibits organoid cell apoptosis, promotes adhesion and proliferation, and reduces cytotoxicity.

[0018] 2. The anticontamination reagent of this invention is compatible with various organ culture media systems. The preparation method is simple and can be directly integrated into the conventional culture process, which significantly improves the success rate of organoid in vitro culture. It has long-term stability. No decay of anticontamination effect was observed after 6 consecutive generations of culture (42 days), and it does not induce microbial resistance. Long-term use does not change the expression of organoid-specific markers and maintains differentiation function.

[0019] 3. The anti-contamination reagent of this invention has low cytotoxicity. At the preferred concentration, the viability of organoid cells is increased by 20-45% compared with the control group without reagent. Y-27632 has a significant anti-apoptotic effect. It has broad-spectrum applicability: it is effective for organoids from various tissues such as intestine, kidney, liver, lung, and mammary gland, and has the ability to salvage contaminated samples.

[0020] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0021] Figure 1 The bar chart shows the comparison of organoid contamination rates under the treatment of the control group (without contamination inhibitor), the conventional antibiotic group (penicillin-streptomycin), and the culture medium containing the contamination inhibitor prepared in Example 2 of this invention.

[0022] Figure 2 The image shows a comparison of organoid cell viability under treatment in the control group (without anticontamination reagent), the conventional antibiotic group (penicillin-streptomycin), and the culture medium containing anticontamination reagent prepared in Example 2 of this invention.

[0023] Figure 3 The images show the morphological observations of organoids after treatment with conventional antibiotics (penicillin-streptomycin) and the culture medium containing anticontamination reagent prepared in Example 2 of this invention.

[0024] Figure 4 This invention relates to mouse intrahepatic cholangiocarcinoma organoids cultured with the highly efficient organoid anticontamination reagent of this invention.

[0025] Figure 5 Human intrahepatic cholangiocarcinoma organoids were cultured continuously with the addition of the highly efficient organoid anticontamination reagent of this invention.

[0026] Figure 6 Human hepatocellular carcinoma organoids were cultured continuously with the high-efficiency organoid anti-contamination reagent of this invention.

[0027] Figure 7 This is a comparison chart of the results of concentration optimization and synergistic effect verification of the anti-pollution reagent components in Experiment Example 4 of this invention. Detailed Implementation

[0028] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0029] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.

[0030] Example 1: Preparation of stock solution for anti-pollution reagent

[0031] Prepare the mother liquors for each component using the following method:

[0032] Primocin stock solution: Take Primocin powder, prepare a 50 mg / mL stock solution with sterile PBS buffer, filter through a 0.22 μm filter membrane for sterilization, dispense 1 mL / tube, and store at -20℃.

[0033] Amphotericin B stock solution: Take amphotericin B powder, prepare a 0.25 mg / mL stock solution with sterile DMSO, filter through a 0.22 μm filter membrane for sterilization, dispense 1 mL / tube, and store at -20℃.

[0034] Y-27632 stock solution: Take Y-27632 powder, prepare a 100mM stock solution with sterile DMSO, filter it through a 0.22μm filter membrane for sterilization, dispense 0.5mL / tube, and store at -80℃.

[0035] Example 2: Preparation of organoid culture medium containing anticontamination reagent

[0036] The mixed solution addition method was used for preparation: the required stock solution volume was calculated according to the final concentrations of Primocin 50 μg / mL, amphotericin B 0.25 μg / mL, and Y-27632 10 μM, and added sequentially to the organoid complete culture medium. Finally, the volume was adjusted to 500 mL with organoid complete culture medium, and the mixture was gently inverted to mix, thus obtaining the composite culture medium containing the anticontamination reagent.

[0037] Example 3: Preparation of organoid culture medium containing anticontamination reagent

[0038] The preparation method adopted by single-liquid addition was as follows: the required volume of stock solution was calculated according to the final concentration of Primocin 20 μg / mL, amphotericin B 1 μg / mL, and Y-27632 20 μM, and added to the organoid complete culture medium respectively. Finally, the volume was adjusted to 500 mL with organoid complete culture medium and gently inverted to mix, thus obtaining the composite culture medium containing the anticontamination reagent.

[0039] Example 4: Preparation of organoid culture medium containing anticontamination reagent

[0040] The mixed solution addition method was used for preparation: the required volume of stock solution was calculated according to the final concentrations of Primocin 70 μg / mL, amphotericin B 0.1 μg / mL, and Y-27632 5 μM, and added sequentially to the organoid complete culture medium. Finally, the volume was adjusted to 500 mL with organoid complete culture medium, and the mixture was gently inverted to mix, thus obtaining the composite culture medium containing the anticontamination reagent.

[0041] Experiment Example 1: Verification of the anti-contamination effect of hepatocellular carcinoma organoid culture

[0042] Hepatocellular carcinoma organoids were seeded into Matrigel, and three groups were established: a control group (without contamination inhibitor), a conventional antibiotic group (penicillin-streptomycin), and a culture medium containing a contamination inhibitor prepared in Example 2 of this invention. All groups were cultured for 7 days at 37°C and 5% CO2. Results showed that bacterial contamination appeared in the control group on day 3, fungal contamination appeared in the conventional antibiotic group on day 5, while no bacterial or fungal contamination was observed in the group of this invention during the 7-day culture period. The organoid formation rate was significantly higher than that of the control group and the conventional antibiotic group, and the organoids exhibited normal morphology and active proliferation.

[0043] Experimental Example 2: Verification of Liver Organoid Cytotoxicity

[0044] Human liver organoids were cultured in media containing different concentrations of anticontamination reagents. A preferred concentration group (Primocin 50 μg / mL, Amphotericin B 0.25 μg / mL, Y-27632 10 μM) and a reagent-free control group were established. Cell viability was assessed after 72 hours of culture. The results showed that the cell viability of the preferred concentration group was significantly higher than that of the reagent-free control group (p < 0.05), indicating that the present invention is non-cytotoxic within the preferred concentration range and can improve cell activity.

[0045] Experiment Example 3: The effect of contamination suppression and remediation on contaminated hepatocellular carcinoma organoids

[0046] Discard the original contaminated culture medium from the culture wells of contaminated hepatocellular carcinoma organoids; add fresh culture medium containing the reagent of Example 2 of this invention, gently rinse and discard, repeat once; add fresh culture medium containing the reagent of Example 2 of this invention again, and continue culturing in a 37°C, 5% CO2 incubator; observe and record the organoid morphology, culture medium turbidity, and contamination status every 24 hours; replace the fresh culture medium containing the reagent of this invention every 48 hours, and continue culturing for 7 days; before the salvage treatment and on days 1, 3, 5, and 7 after the treatment, collect culture medium for microbial culture identification, and perform organoid cell viability testing (CCK-8 assay) and morphological observation on day 7. On day 7, the organoids proliferated actively, and the cell viability was significantly higher than the other two groups, indicating that the contamination inhibitor of this invention has a good salvage effect on contaminated hepatocellular carcinoma organoids.

[0047] Experiment Example 4: Optimization of Concentration of Anti-pollution Reagent Components and Verification of Synergistic Effect

[0048] To determine the optimal ratio and synergistic effect of Primocin, amphotericin B, and Y-27632, combinations of anticontamination reagents with different concentration gradients were set up, and the anticontamination effect and cell viability were tested in a hepatocellular carcinoma organoid culture system.

[0049] Experimental Design:

[0050] Human hepatocyte carcinoma organoids were cultured in basal medium (Advanced DMEM / F12 + 1× B27 + 10 mM Hepes) and the treatment groups were set up as shown in Table 1 below (final concentration of each component):

[0051] Table 1

[0052]

[0053] Each group consisted of 3 replicates, and the cells were cultured at 37°C and 5% CO2 for 7 days. Daily observations were made of contamination (turbidity of the culture medium, pH changes, and the presence of hyphae or bacteria under a microscope). On day 7, organoid cell viability was assessed using the CCK-8 assay.

[0054] result:

[0055] Group I (control) showed bacterial or fungal contamination on days 2-3.

[0056] Group B (Primocin alone) inhibited bacteria, but fungal contamination appeared on day 4.

[0057] Group C (amycin B alone) can inhibit fungi, but bacterial contamination occurs on day 3.

[0058] Group D (Primocin + Amphotericin B) showed no obvious contamination within 7 days, but cell viability was lower than that of Group A, suggesting that the lack of Y-27632 led to impaired cell proliferation.

[0059] Groups E and F had narrow anti-pollution spectrums (lacking antifungal or antibacterial components, respectively), and both showed specific types of contamination on days 4-5.

[0060] Group A (Preferred Formula): No contamination within 7 days, highest cell viability.

[0061] Both groups G and H were free of contamination, but the cell viability of group G (low concentration) was not significantly different from that of group A, while the cell viability of group H (high concentration) was slightly lower than that of group A, indicating that the optimal concentration achieves the best balance between contamination suppression and cell safety.

[0062] Experiment Example 5: Verification of the pollution suppression effect of different organoid types

[0063] The anticontamination reagent (preferred concentration) of Example 2 of the present invention was added to the culture medium of organoids from different sources to verify its broad-spectrum applicability.

[0064] Experimental subjects:

[0065] Mouse intestinal organoids, human kidney organoids, human lung adenocarcinoma organoids, and human breast cancer organoids.

[0066] Methods: The above-mentioned organoid culture systems were established separately. Each system had a control group (without anti-contamination reagent) and an experimental group (with anti-contamination reagent of Example 2 of this invention added). Each group had 5 independent culture wells and were cultured continuously for 10 days. The time of contamination and organoid formation efficiency were recorded.

[0067] The results are shown in Table 2 below:

[0068] Table 2

[0069]

[0070] As shown in Table 2 above, no bacterial or fungal contamination was observed in any of the experimental groups during the 10-day culture period. The organoids exhibited normal morphology, active proliferation, and a formation rate exceeding 95%, indicating that the contamination inhibitor of this invention has good compatibility and contamination inhibition effect on organoids from different tissue sources.

[0071] This invention achieves dual and highly efficient inhibition of bacteria and fungi through the synergistic effect of three components. At the same time, Y-27632 can inhibit organoid cell apoptosis, promote adhesion and proliferation, significantly reduce cytotoxicity, adapt to various organoid culture media systems, and is easy to operate. The highly efficient organoid anticontamination reagent provided by this invention can achieve broad-spectrum and stable anticontamination effect while ensuring the safety of organoid cells, significantly improving the success rate of organoid culture, and is suitable for various organoid culture systems.

[0072] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0073] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A highly efficient organoid anti-contamination agent, characterized in that, It consists of Primocin, amphotericin B, and Y-27632.

2. The highly efficient organoid anti-contamination reagent as described in claim 1, characterized in that: In organoid culture systems, the final concentrations of Primocin were 20-70 μg / mL, Amphotericin B was 0.1-1 μg / mL, and Y-27632 was 5-20 μM.

3. The highly efficient organoid anti-contamination reagent as described in claim 2, characterized in that: In the organoid culture system, the final concentration of Primocin was 50 μg / mL, the final concentration of amphotericin B was 0.25 μg / mL, and the final concentration of Y-27632 was 10 μM.

4. The highly efficient organoid anti-contamination reagent as described in claim 4, characterized in that: The anti-pollution reagent is added in the form of a mother liquor, wherein: Primocin stock solution was prepared using sterile PBS buffer, with a stock solution concentration of 50 mg / mL; The stock solution of amphotericin B was prepared using sterile DMSO, with a concentration of 0.25 mg / mL. Y-27632 stock solution was prepared using sterile DMSO with a concentration of 100 mM.

5. The highly efficient organoid anti-contamination reagent as described in claim 1, characterized in that: Organoids include intestinal organoids, liver organoids, kidney organoids, lung organoids, intrahepatic bile duct carcinoma organoids, hepatocellular carcinoma organoids, and lung cancer organoids.

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