Culture method and application of patient-derived laryngeal cancer organoid

Abstract

The invention belongs to the field of cell culture, and particularly relates to a culture method of a patient-derived laryngeal cancer organ, the culture method comprises sample treatment and seed plate culture: the sample treatment comprises the following steps: S1, sample cleaning: transferring a sample to a 15ml centrifugal tube, adding 5ml of a PBS solution containing 3% of three antibodies, cleaning until no obvious turbidity exists, discarding supernatant, and repeatedly cleaning for 5-10 times; s2, observing and recording the sample: transferring the cleaned sample into a 60 mm culture dish, and photographing and recording the state of the sample, the method provided by the invention achieves the effect of fully utilizing the obtained sample to obtain an enough number of organoids with good activity by establishing a culture, passage, cryopreservation and recovery method of the organoids capable of retaining the immune microenvironment, improves the success rate of primary modeling, and reduces the cost. The method maximizes the acquisition rate of the patient-derived laryngocarcinoma organs, and provides a quicker and better experimental model for the fields of tumor pathogenesis research, drug screening, drug toxicity, pharmacodynamic tests and individualized precision medical treatment.

Description

Technical Field

[0001] This invention belongs to the field of cell culture, specifically a method and application for culturing laryngeal cancer organoids derived from patients. Background Technology

[0002] As an emerging in vitro three-dimensional cell culture technology, organoids can form "micro-tissues" that retain tissue structure and biological information. They can simulate various cell types, structures and functional characteristics of organs in vitro and have been widely used in fields such as stem cell fate regulation, developmental aging research, genetic disease and tumor model construction. In particular, patient-derived organoid culture systems combine the advantages of heritable manipulation of two-dimensional cell lines with the complex three-dimensional system characteristics of human tumor tissue xenograft models. They not only preserve the key histopathological, genetic, and phenotypic characteristics of the maternal tumor, but also maintain the heterogeneity of the source tumor. Moreover, they are closer to the real in vivo state than traditional two-dimensional cell culture, and are more suitable for high-throughput and low-time cost requirements than animal models. The morphology and scale of individuals are basically uniform, providing an excellent experimental model for tumor pathogenesis research, drug screening, and personalized precision medicine. Its research began in 2009 when Sato et al. used LGR5+ intestinal stem cells to establish three-dimensional epithelial organoids. Subsequently, scholars have successfully cultured organoid tissues of various organs such as the colon, pancreas, and stomach based on this.

[0003] However, current organoid research has two major limitations: First, research on the culture of laryngeal cancer organoids is relatively scarce and lags significantly behind research on other mature organoids. Secondly, while existing organoid culture systems can maintain the stability and three-dimensional structure of tumor cells well, they are difficult to retain other cell types such as immune cells in tumor tissues. This results in organoids having defects in maintaining tumor heterogeneity and cell diversity, which limits their application scope to a certain extent.

[0004] Therefore, the present invention provides a method for culturing patient-derived laryngeal cancer organoids and their application. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, at least one technical problem raised in the background art is solved.

[0006] The technical solution adopted by this invention to solve its technical problem is: a method for culturing patient-derived laryngeal cancer organoids, comprising sample processing and seed plate culture. Sample processing includes the following steps: S1. Sample cleaning: Transfer the sample to a 15ml centrifuge tube, add 5ml of PBS solution containing 3% triple antibody and wash until there is no obvious turbidity. Discard the supernatant and repeat the washing 5-10 times. S2. Sample observation and recording: Transfer the cleaned sample into a 60mm culture dish, take photos to record the sample status, observe and remove obvious adipose tissue, blood vessels and damaged tissue from the sample. S3. Sample cutting: Add 50ul of a solution containing 2.5mM2ml EDTA to the tissue block and cut the tissue into small pieces or a paste with a sterile blade. S4. Tissue digestion: After adding culture medium and mixing by pipetting, transfer the cut tissue to a 15ml centrifuge tube, add 2-3ml of digestion solution and digest for 3 minutes, add PBS to stop digestion, centrifuge at 300g / 3min and discard the supernatant. S5. Digestive enzyme removal: Add an appropriate amount of PBS to wash the tissue, centrifuge at 300g / min, discard the supernatant, and repeat this step twice to remove digestive enzymes. S6. Grinding and filtration: Cut off the 1ml pipette tip and aspirate the tissue block and liquid onto a 100μm cell mesh screen. Use the plunger of a 5ml syringe to grind and filter the tissue on the screen until the tissue on the screen no longer shrinks. S7. Centrifugation of suspension: Transfer all the ground and filtered laryngeal cancer cell suspension to a 15ml centrifuge tube, centrifuge at 300g / 3min, and discard the supernatant. S8. Matrix gel resuspending: Add 2 times the volume of matrix gel to resuspend the cell suspension. The entire process is carried out under ice bath conditions. The pipetting process is gentle to avoid generating air bubbles. Seed culture includes the following steps: S9. Inoculating droplets: Take 50 μL of cell suspension and inoculate it into a preheated 24-well cell culture plate. Fix the gel at 37°C for 30 min. S10. Culture medium addition: After the gel is solidified, slowly add 500 μL of organoid culture medium to each well, take pictures under a microscope to record the morphology of the primary organoids, and then place the culture medium in an incubator at 37°C and 5% CO2 for incubation. S11. Routine maintenance: Change the culture medium every three days. Observe the growth and density of organoids after one week. Decide whether to passage based on the observation results. The culture process should always be carried out in an incubator at 37°C and 5% CO2.

[0007] Application of a culture method for patient-derived laryngeal cancer organoids, the application of which includes organoid passage and cryopreservation: Organoid passage includes the following steps: S1.1 When the size of laryngeal cancer organoids exceeds 200μm or the number exceeds 20 under a 10x microscope, passage is performed. Prepare pre-cooled matrix gel, PBS, low-absorption pipette tips, 15ml centrifuge tubes, 6-well plates, and culture medium, digestive enzymes, and 24-well plates preheated to 37℃ in advance. S1.2 Remove the 24-well plate from the incubator, aspirate the culture medium from the wells, and avoid aspirating or touching the gel droplets inside the wells; add pre-cooled PBS solution along the well wall, and gently blow with a pre-cooled 1ml pipette tip with low adsorption to dissolve the gel; after the gel droplets in the wells separate from the plate, transfer them together with the PBS solution to a 15ml centrifuge tube, place the centrifuge tube in a -20℃ freezer for 5min or a 4℃ freezer for 4min until the matrix gel is completely dissolved, centrifuge at 300g / 5min, and discard the supernatant; S1.3. Depending on the morphology of the organoids in the sample, use pipette to agitate or enzymatically digest them. For thin-walled, cystic laryngeal cancer organoids, slowly agitate them 20-30 times in a centrifuge tube. For compact, dense organoids, briefly digest them with preheated digestive enzymes for 2-3 minutes, then agitate them 5-10 times. Neither mechanical nor enzymatic methods should involve vigorous agitation to avoid mechanical damage to the organoid structure. Collect all liquids into 15ml centrifuge tubes, centrifuge at 300g / 5min, discard the supernatant, and collect the organoid precipitate. S1.4. Based on the organoid precipitate, calculate the number of planting wells according to a passage ratio of 1:2-1:3; according to the standard that the total volume of organoid suspension and Matrigel per well is 30 μl, the ratio of the two is 1:2, and the culture density is 30-50 organoids / μl, add basal culture medium and Matrigel to the organoid precipitate and mix by pipetting; while pipetting the mixture, add 30 μl vertically to the middle of the 24-well plate; place the 24-well plate in a 37℃ incubator for 30 min to allow the gel to solidify; S1.5 After the gel solidifies and forms stable droplets, add 500 μl of preheated organoid culture medium to each well; take pictures under a microscope to record the morphology and generation of the organoids, and then incubate them in a 37℃, 5% CO2 incubator.

[0008] Application of a method for culturing patient-derived laryngeal cancer organoids, with organoid cryopreservation including the following steps: S2.1 When the density reaches the requirement during subculturing, it can be prepared for cryopreservation; it is necessary to thaw the gel first, and the specific steps for thawing are the same as those in step S1.2. S2.2 After gelation, transfer the liquid to a 15ml centrifuge tube, rinse the bottom of the 24-well plate 2-3 times with 2ml of PBS solution; centrifuge at 300g / 5min and discard the supernatant; S2.3. After resuspending with an appropriate amount of PBS solution, gently pipette to mix; calculate the volume of the frozen solution and the number of dispensing tubes, then centrifuge at 400g / 5min and discard the supernatant. S2.4. Add the corresponding volume of cryopreservation solution according to the counting results, gently pipette to mix the organoids, and dispense 1 ml into each cryopreservation tube. S2.5 Set the program control of the programmable cooling instrument to decrease the temperature by 1℃ per minute; when the temperature drops to -80℃, transfer the cryopreservation tubes into liquid nitrogen for long-term storage.

[0009] The beneficial effects of this invention are as follows: The present invention describes a method and application for culturing patient-derived laryngeal cancer organoids. The method achieves sample purification by washing with PBS containing 3% triple antibodies 5-10 times and removing non-tumor components such as adipose tissue. It is combined with 3-minute short-time enzymatic digestion and gentle grinding and filtration to reduce cell loss, thus solving the problem of scarce patient laryngeal cancer samples. At the same time, key parameters such as 300g centrifugation force, ice bath matrix gel operation, and 37℃ / 5% CO2 culture environment are controlled throughout the process. Furthermore, the method combines morphological passage to avoid organoid damage, which greatly improves the success rate of primary modeling and reduces the cost of experimental repetition. Secondly, by using matrix gel to construct a three-dimensional microenvironment to simulate in vivo growth conditions, and with medium changes every 3 days and a standardized passage density of 30-50 organs / μl, we can ensure that organoids maintain high activity and morphology and function that are highly consistent with the original tumor during long-term culture. Furthermore, by using a gentle treatment process such as low-adsorption pipette tip gelation and avoiding vigorous blowing, we can preserve the original immune microenvironment, making the organoids more closely resemble the real state of tumors in vivo. The entire process is clear and controllable, such as programmed cooling at 1℃ / min during cryopreservation and passage ratio of 1:2-1:3, which lowers the operational threshold and facilitates laboratory reproducibility. Furthermore, it can achieve long-term stable preservation of organoids through programmed cooling and liquid nitrogen cryopreservation, without relying on fresh samples, and can be revived and used at any time. To establish methods for culturing, passaged, cryopreserved, and thawed organoids that preserve the immune microenvironment, thereby achieving the goal of fully utilizing the obtained samples to obtain a sufficient number of highly viable organoids, improving the success rate of primary modeling, maximizing the acquisition rate of patient-derived laryngeal cancer organoids, and providing faster and better experimental models for tumor pathogenesis research, drug screening, drug toxicity, efficacy testing, and personalized precision medicine. Detailed Implementation

[0010] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0011] This invention discloses a method and application for culturing patient-derived laryngeal cancer organoids, the method comprising sample processing and seed plate culture: Sample processing includes the following steps: S1. Sample cleaning: Transfer the sample to a 15ml centrifuge tube, add 5ml of PBS solution containing 3% triple antibody and wash until there is no obvious turbidity. Discard the supernatant and repeat the washing 5-10 times. S2. Sample observation and recording: Transfer the cleaned sample into a 60mm culture dish, take photos to record the sample status, observe and remove obvious adipose tissue, blood vessels and damaged tissue from the sample. S3. Sample cutting: Add 50ul of a solution containing 2.5mM2ml EDTA to the tissue block and cut the tissue into small pieces or a paste with a sterile blade. S4. Tissue digestion: After adding culture medium and mixing by pipetting, transfer the cut tissue to a 15ml centrifuge tube, add 2-3ml of digestion solution and digest for 3 minutes, add PBS to stop digestion, centrifuge at 300g / 3min and discard the supernatant. S5. Digestive enzyme removal: Add an appropriate amount of PBS to wash the tissue, centrifuge at 300g / min, discard the supernatant, and repeat this step twice to remove digestive enzymes. S6. Grinding and filtration: Cut off the 1ml pipette tip and aspirate the tissue block and liquid onto a 100μm cell mesh screen. Use the plunger of a 5ml syringe to grind and filter the tissue on the screen until the tissue on the screen no longer shrinks. S7. Centrifugation of suspension: Transfer all the ground and filtered laryngeal cancer cell suspension to a 15ml centrifuge tube, centrifuge at 300g / 3min, and discard the supernatant. S8. Matrix gel resuspending: Add 2 times the volume of matrix gel to resuspend the cell suspension. The entire process is carried out under ice bath conditions. The pipetting process is gentle to avoid generating air bubbles. Seed culture includes the following steps: S9. Inoculating droplets: Take 50 μL of cell suspension and inoculate it into a preheated 24-well cell culture plate. Fix the gel at 37°C for 30 min. S10. Culture medium addition: After the gel is solidified, slowly add 500 μL of organoid culture medium to each well, take pictures under a microscope to record the morphology of the primary organoids, and then place the culture medium in an incubator at 37°C and 5% CO2 for incubation. S11. Routine maintenance: Change the culture medium every three days. Observe the growth and density of organoids after one week. Decide whether to passage based on the observation results. The culture process should always be carried out in an incubator at 37°C and 5% CO2.

[0012] Application of a culture method for patient-derived laryngeal cancer organoids, the application of which includes organoid passage and cryopreservation: Organoid passage includes the following steps: S1.1 When the size of laryngeal cancer organoids exceeds 200μm or the number exceeds 20 under a 10x microscope, passage is performed. Prepare pre-cooled matrix gel, PBS, low-absorption pipette tips, 15ml centrifuge tubes, 6-well plates, and culture medium, digestive enzymes, and 24-well plates preheated to 37℃ in advance. S1.2 Remove the 24-well plate from the incubator, aspirate the culture medium from the wells, and avoid aspirating or touching the gel droplets inside the wells; add pre-cooled PBS solution along the well wall, and gently blow with a pre-cooled 1ml pipette tip with low adsorption to dissolve the gel; after the gel droplets in the wells separate from the plate, transfer them together with the PBS solution to a 15ml centrifuge tube, place the centrifuge tube in a -20℃ freezer for 5min or a 4℃ freezer for 4min until the matrix gel is completely dissolved, centrifuge at 300g / 5min, and discard the supernatant; S1.3. Depending on the morphology of the organoids in the sample, use pipette to agitate or enzymatically digest them. For thin-walled, cystic laryngeal cancer organoids, slowly agitate them 20-30 times in a centrifuge tube. For compact, dense organoids, briefly digest them with preheated digestive enzymes for 2-3 minutes, then agitate them 5-10 times. Neither mechanical nor enzymatic methods should involve vigorous agitation to avoid mechanical damage to the organoid structure. Collect all liquids into 15ml centrifuge tubes, centrifuge at 300g / 5min, discard the supernatant, and collect the organoid precipitate. S1.4. Based on the organoid precipitate, calculate the number of planting wells according to a passage ratio of 1:2-1:3; according to the standard that the total volume of organoid suspension and Matrigel per well is 30 μl, the ratio of the two is 1:2, and the culture density is 30-50 organoids / μl, add basal culture medium and Matrigel to the organoid precipitate and mix by pipetting; while pipetting the mixture, add 30 μl vertically to the middle of the 24-well plate; place the 24-well plate in a 37℃ incubator for 30 min to allow the gel to solidify; S1.5 After the gel solidifies and forms stable droplets, add 500 μl of preheated organoid culture medium to each well; take pictures under a microscope to record the morphology and generation of the organoids, and then incubate them in a 37℃, 5% CO2 incubator.

[0013] Application of a method for culturing patient-derived laryngeal cancer organoids, with organoid cryopreservation including the following steps: S2.1 When the density reaches the requirement during subculturing, it can be prepared for cryopreservation; it is necessary to thaw the gel first, and the specific steps for thawing are the same as those in step S1.2. S2.2 After gelation, transfer the liquid to a 15ml centrifuge tube, rinse the bottom of the 24-well plate 2-3 times with 2ml of PBS solution; centrifuge at 300g / 5min and discard the supernatant; S2.3. After resuspending with an appropriate amount of PBS solution, gently pipette to mix; calculate the volume of the frozen solution and the number of dispensing tubes, then centrifuge at 400g / 5min and discard the supernatant. S2.4. Add the corresponding volume of cryopreservation solution according to the counting results, gently pipette to mix the organoids, and dispense 1 ml into each cryopreservation tube. S2.5 Set the program control of the programmable cooling instrument to decrease the temperature by 1℃ per minute; when the temperature drops to -80℃, transfer the cryopreservation tubes into liquid nitrogen for long-term storage.

[0014] Specifically, This protocol establishes a method for culturing, passaged, cryopreserving, and thawing organoids that preserve the immune microenvironment. This method aims to fully utilize the obtained samples to acquire a sufficient number of highly viable organoids, improve the success rate of primary modeling, maximize the acquisition rate of patient-derived laryngeal cancer organoids, and provide a faster and better experimental model for tumor pathogenesis research, drug screening, drug toxicity, efficacy testing, and personalized precision medicine.

[0015] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for culturing patient-derived laryngeal cancer organoids, characterized in that, This culture method includes sample processing and seed plate culture: The sample processing includes the following steps: S1. Sample cleaning: Transfer the sample to a 15ml centrifuge tube, add 5ml of PBS solution containing 3% triple antibody and wash until there is no obvious turbidity. Discard the supernatant and repeat the washing 5-10 times. S2. Sample observation and recording: Transfer the cleaned sample into a 60mm culture dish, take photos to record the sample status, observe and remove obvious adipose tissue, blood vessels and damaged tissue from the sample. S3. Sample cutting: Add 50ul of a solution containing 2.5mM2ml EDTA to the tissue block and cut the tissue into small pieces or a paste with a sterile blade. S4. Tissue digestion: After adding culture medium and mixing by pipetting, transfer the cut tissue to a 15ml centrifuge tube, add 2-3ml of digestion solution and digest for 3 minutes, add PBS to stop digestion, centrifuge at 300g / 3min and discard the supernatant. S5. Digestive enzyme removal: Add an appropriate amount of PBS to wash the tissue, centrifuge at 300g / min, discard the supernatant, and repeat this step twice to remove digestive enzymes. S6. Grinding and filtration: Cut off the 1ml pipette tip and aspirate the tissue block and liquid onto a 100μm cell mesh screen. Use the plunger of a 5ml syringe to grind and filter the tissue on the screen until the tissue on the screen no longer shrinks. S7. Centrifugation of suspension: Transfer all the ground and filtered laryngeal cancer cell suspension to a 15ml centrifuge tube, centrifuge at 300g / 3min, and discard the supernatant. S8. Matrix gel resuspending: Add 2 times the volume of matrix gel to resuspend the cell suspension. The entire process is carried out under ice bath conditions. The pipetting process is gentle to avoid generating air bubbles. The seed plate culture includes the following steps: S9. Inoculating droplets: Take 50 μL of cell suspension and inoculate it into a preheated 24-well cell culture plate. Fix the gel at 37°C for 30 min. S10. Culture medium addition: After the gel is solidified, slowly add 500 μL of organoid culture medium to each well, take pictures under a microscope to record the morphology of the primary organoids, and then place the culture medium in an incubator at 37°C and 5% CO2 for incubation. S11. Routine maintenance: Change the culture medium every three days. Observe the growth and density of organoids after one week. Decide whether to passage based on the observation results. The culture process should always be carried out in an incubator at 37°C and 5% CO2.

2. The application of a method for culturing patient-derived laryngeal cancer organoids, wherein the method is used in accordance with the method for culturing patient-derived laryngeal cancer organoids as described in claim 1, characterized in that, This culture method has applications including organoid passage and organoid cryopreservation. The organoid passage includes the following steps: S1.1 When the size of laryngeal cancer organoids exceeds 200μm or the number exceeds 20 under a 10x microscope, passage is performed. Prepare pre-cooled matrix gel, PBS, low-absorption pipette tips, 15ml centrifuge tubes, 6-well plates, and culture medium, digestive enzymes, and 24-well plates preheated to 37℃ in advance. S1.2 Remove the 24-well plate from the incubator, aspirate the culture medium from the wells, and avoid aspirating or touching the gel droplets inside the wells; add pre-cooled PBS solution along the well wall, and gently blow with a pre-cooled 1ml pipette tip with low adsorption to dissolve the gel; after the gel droplets in the wells separate from the plate, transfer them together with the PBS solution to a 15ml centrifuge tube, place the centrifuge tube in a -20℃ freezer for 5min or a 4℃ freezer for 4min until the matrix gel is completely dissolved, centrifuge at 300g / 5min, and discard the supernatant; S1.

3. Depending on the morphology of the organoids in the sample, use pipette to agitate or enzymatically digest them. For thin-walled, cystic laryngeal cancer organoids, slowly agitate them 20-30 times in a centrifuge tube. For compact, dense organoids, briefly digest them with preheated digestive enzymes for 2-3 minutes, then agitate them 5-10 times. Neither mechanical nor enzymatic methods should involve vigorous agitation to avoid mechanical damage to the organoid structure. Collect all liquids into 15ml centrifuge tubes, centrifuge at 300g / 5min, discard the supernatant, and collect the organoid precipitate. S1.

4. Based on the organoid precipitate, calculate the number of planting wells according to a passage ratio of 1:2-1:3; according to the standard that the total volume of organoid suspension and Matrigel per well is 30 μl, the ratio of the two is 1:2, and the culture density is 30-50 organoids / μl, add basal culture medium and Matrigel to the organoid precipitate and mix by pipetting; while pipetting the mixture, add 30 μl vertically to the middle of the 24-well plate; place the 24-well plate in a 37℃ incubator for 30 min to allow the gel to solidify; S1.5 After the gel solidifies and forms stable droplets, add 500 μl of preheated organoid culture medium to each well; take pictures under a microscope to record the morphology and generation of the organoids, and then incubate them in a 37℃, 5% CO2 incubator.

3. The application of the method for culturing patient-derived laryngeal cancer organoids according to claim 2, characterized in that: The cryopreservation of the organoids includes the following steps: S2.1 When the density reaches the requirement during subculturing, it can be prepared for cryopreservation; it is necessary to thaw the gel first, and the specific steps for thawing are the same as those in step S1.

2. S2.2 After gelation, transfer the liquid to a 15ml centrifuge tube, rinse the bottom of the 24-well plate 2-3 times with 2ml of PBS solution; centrifuge at 300g / 5min and discard the supernatant; S2.

3. After resuspending with an appropriate amount of PBS solution, gently pipette to mix; calculate the volume of the frozen solution and the number of dispensing tubes, then centrifuge at 400g / 5min and discard the supernatant. S2.

4. Add the corresponding volume of cryopreservation solution according to the counting results, gently pipette to mix the organoids, and dispense 1 ml into each cryopreservation tube. S2.5 Set the program control of the programmable cooling instrument to decrease the temperature by 1℃ per minute; when the temperature drops to -80℃, transfer the cryopreservation tubes into liquid nitrogen for long-term storage.