Method for culturing lung cancer tumor organoid

Abstract

The invention relates to a method for culturing lung cancer tumor organoid. The method sequentially comprises the following steps: (1) selecting non-necrotic effective tissues from human lung cancer tissues as lung cancer tissue samples; (2) cleaning the collected tissue sample; (3) evaluating the tissue sample, removing non-epithelial components, and removing obviously necrotic tissue components; (4) selecting a tissue digestive juice digestion and / or mechanical disruption method to obtain cell clusters or single cells; (5) carrying out cell precipitation resuspension and three-dimensional culture inoculation; and (6) culturing the organoid. According to the culture method, a lung cancer organoid model highly similar to a source tumor tissue in the aspects of cell characteristics, pathological histological characteristics, molecular characteristics and the like can be efficiently cultured, so that the organoid can be successfully applied to the fields of lung cancer disease research, drug development, individualized treatment and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a method for culturing lung cancer tumor organoids. Background Art

[0002] Currently, 2D cell lines are still the most commonly used in vitro models in cancer research. However, lung cancer cell lines have some significant limitations. First of all, it cannot reflect individual tumor characteristics in real time, and the success rate of establishing primary lung cancer is less than 5%. Human-derived tumor organoids have a near-physiological structure and retain the specific functions of natural tumors. The tissue structure, cell types, and gene characteristics of tumor organoids are highly consistent with the tumors of the patients themselves. Therefore, organoid technology can significantly improve drug screening and generalize drug responses. Organoids from lung cancer patients can be used for further high-throughput drug screening, and can be used as surrogates to test drugs for patients, truly achieving "precision medicine". Through organoid technology, 5-15 drugs can be tested for patients within 14 days, increasing the treatment benefit rate of tumor patients from 30% to over 70%. At the same time, organoid culture also has advantages such as a shorter experimental period and lower costs.

[0003] On November 30, 2021, the Center for Drug Evaluation of the National Medical Products Administration issued the "Technical Guidelines for Non-Clinical Research and Evaluation of Gene Therapy Products (Trial)" and the "Technical Guidelines for Non-Clinical Research of Gene-Modified Cell Therapy Products (Trial)", and for the first time included organoids in the guidelines for gene therapy and gene-modified cell therapy products. Organoid culture technology: Researchers extract the main components such as tumor stem cells and stromal cells from lung cancer tissues derived from patients, and through culture, provide extracellular matrix, nutrients, and various signal regulatory factors in vitro to enable the growth of organoids in vitro, and can simulate the lung cancer growth environment in vitro. Currently, lung cancer organoids are gradually being applied in the fields of lung cancer disease research, drug development, and personalized treatment. However, there are currently problems with the culture of lung cancer organoids, such as non-standardized culture operation procedures and unclear culture protocols, which ultimately lead to inconsistent culture effects. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for culturing lung cancer tumor organoids, which can efficiently culture a lung cancer organoid model that is highly similar to the source tumor tissue in terms of cell characteristics, histopathological characteristics, and molecular characteristics, so as to successfully apply organoids in the fields of lung cancer disease research, drug development, and personalized treatment.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A method for culturing lung cancer tumor organoids, characterized by successively including the following steps:

[0007] (1) Select non-necrotic effective tissue from human lung cancer tissue as a lung cancer tissue sample;

[0008] (2) Wash the collected tissue sample;

[0009] (3) Evaluate the tissue sample, remove non-epithelial components, and remove significantly necrotic tissue components;

[0010] (4) Select a tissue digestive solution for digestion and / or a mechanical fragmentation method to obtain cell clusters or single cells;

[0011] (5) Resuspend the cell precipitate and inoculate for three-dimensional culture

[0012] Take a scaffold material to resuspend the cell precipitate, mix well and inoculate, or resuspend with a lung cancer organoid culture medium and then inoculate. After the scaffold material solidifies, add a lung cancer organoid culture medium for culture;

[0013] (6) Organoid culture.

[0014] For further clarification, the above-mentioned method for culturing lung cancer tumor organoids is characterized by sequentially including the following steps:

[0015] (1) Acquisition of tissue sample

[0016] Select non-necrotic effective tissue from human lung cancer tissue obtained by conventional means such as puncture or surgery as a lung cancer tissue sample. Immerse the collected lung cancer tissue sample completely in sterile medical physiological saline and store it under low temperature conditions of 0°C to 4°C, and process the lung cancer tissue sample in a timely manner within 0 to 8 hours;

[0017] (2) Pretreatment of tissue sample

[0018] Wash the collected lung cancer tissue sample with a pre-cooled 4°C tissue washing solution. When washing, shake it quickly up, down, left, and right with moderate shaking strength until the washing solution is clear;

[0019] (3) Evaluation of tissue sample

[0020] Evaluate the content of epithelial cells in the obtained lung cancer tissue sample, and use ophthalmic scissors and forceps to remove non-epithelial components as much as possible, including muscle and adipose tissue, and remove significantly necrotic tissue components;

[0021] (4) Digestion of tissue mass

[0022] Select a tissue digestive solution for digestion and / or a mechanical fragmentation method to obtain cell clusters or single cells under the condition of 37°C; the tissue digestive solution should ensure sterility, and the digestion time should be controlled within the range of 0 to 2 hours, not too long, and should be judged according to the size and degree of deterioration of the tissue mass;

[0023] (5) Resuspension of cell pellet and seeding for three-dimensional culture

[0024] Take an appropriate amount of scaffold material to resuspend the cell pellet. After mixing evenly, seed it in a culture well plate, or resuspend it with lung cancer organoid medium and then seed it on a 24-well plate. Wait for the scaffold material to solidify, add lung cancer organoid medium to cover the cells, and culture it in a 37 °C carbon dioxide cell incubator;

[0025] (6) Organoid culture

[0026] According to the growth of organoids, change the lung cancer organoid medium once every 3 to 5 days.

[0027] For further clarification, the above tissue washing solution consists of the following components: 50 ml of phosphate buffered saline (PBS), 1x of Penicillin / Streptomycin, and 1x of Primocin antibiotic.

[0028] For further clarification, the above tissue digestion solution consists of the following components: 50 ml of Advance DMEM / F12 basal medium, 2 - 10 μM of ROCK inhibitor (ROCK inhibitor Y-27632), and 200 - 1000 U / mL or 2 - 5 mg / mL of type IV collagenase (IV Collagenase).

[0029] For further clarification, the above scaffold material is Matrigel. Currently, the commonly used 3D scaffold material for organoid culture is Matrigel, which is extracted from the extracellular matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and mainly consists of laminin and type IV collagen, providing structural support and signaling factors similar to the extracellular environment.

[0030] For further clarification, the above-mentioned lung cancer organoid culture medium consists of the following components: 50 ml of Advance DMEM / F12 basal medium, 1x Glutamax, 1x 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1x B27 supplement, 1x Primocin antibiotic, 1x Penicillin / Streptomycin, 2 - 5 mM of N-acetylcysteine, 2 - 10 μM of ROCK inhibitor Y-27632, 50 - 250 ng / ml of human Noggin, 0.1 - 2 μM of WNT signaling pathway activator R-Spondin 1, 50 - 250 ng / ml of human epidermal growth factor (EGF), 2 - 5 mM of Sirtuins inhibitor Nicotinamide, 0.1 - 2 μM of TGFβ inhibitor A83-01, 50 - 250 ng / ml of fibroblast growth factor 10 (FGF10), 50 - 250 ng / ml of recombinant human keratinocyte growth factor (FGF 7), and 0.1 - 2 μM of P38MAPK inhibitor SB202190.

[0031] The present invention has the following beneficial effects:

[0032] The present invention provides a method for culturing lung cancer tumor organoids, which efficiently cultivates a lung cancer organoid model that is highly similar to the source tumor tissue in terms of cell characteristics, histopathological features, and molecular features. Thus, the organoids are successfully applied to the fields of lung cancer disease research, drug development, and personalized treatment. It uses tumor specimens, biopsy tissues, or liquid biopsy samples removed by surgical resection. After processing, tumor organoids mainly in the form of three-dimensional multicellular aggregates are cultured under specific culture conditions, which can reproduce the pathological characteristics of patients, can simulate the organizational structure and function in the patient's body to the greatest extent, and can be subcultured stably for a long time. Compared with the prior art, the present invention has the characteristics of being highly similar to patient tissues compared with cell lines, and the organoid culture process is more efficient and standardized, greatly improving the efficiency of culturing lung cancer organoids. The method of the present invention establishes a unified operation procedure and requirements for culturing human-derived lung cancer organoids, further standardizes the culturing technology of human-derived lung cancer organoids in the scientific research and biopharmaceutical industries, reduces the deviation of culture results and subsequent research and application outputs caused by different experimental operations, and thus promotes the application of human-derived lung cancer organoids in the biopharmaceutical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1It is the morphological observation diagram of the lung cancer organoid in Example 1 of the present invention under a 4X microscope optical microscope.

[0034] Figure 2 It is the morphological observation diagram of the lung cancer organoid in Example 1 of the present invention under a 10X microscope optical microscope.

[0035] Figure 3 It is the morphological observation diagram of the lung cancer organoid in Example 1 of the present invention under a 20X microscope optical microscope.

[0036] Figure 4 It is the morphological observation diagram of the lung cancer organoid in Example 1 of the present invention under a 40X microscope optical microscope.

[0037] Figure 5 It is the histological feature HE staining observation diagram of the lung cancer organoid in Example 1 of the present invention.

[0038] Figure 6 It is the histological feature HE staining observation diagram of the lung cancer organoid in Example 1 of the present invention.

[0039] Figure 7 It is the histological feature HE staining observation diagram of the lung cancer organoid in Example 1 of the present invention.

[0040] Figure 8 It is the histological feature HE staining observation diagram of the lung cancer organoid in Example 1 of the present invention.

[0041] Figure 9 It is the immunofluorescence staining pathological marker expression diagram of the lung cancer organoid in Example 1 of the present invention.

[0042] Figure 10 It is the immunofluorescence staining pathological marker expression diagram of the lung cancer organoid in Example 1 of the present invention. Detailed implementation manners

[0043] In order to be able to understand the features and technical content of the embodiments of the present application in more detail, the implementation of the embodiments of the present application will be described in detail below with reference to the accompanying drawings. The accompanying drawings are only for reference and illustration purposes and are not used to limit the embodiments of the present application.

[0044] Example 1

[0045] A culture method for a lung cancer tumor organoid sequentially includes the following steps:

[0046] (1) Acquisition of tissue samples

[0047] Human lung cancer tissues are obtained through conventional surgical methods. The doctor screens out the non-necrotic effective tissues as lung cancer tissue samples according to indicators such as the degree of deterioration and activity. The collected lung cancer tissue samples are completely immersed in sterile medical physiological saline and stored at 4°C under low-temperature conditions. And the lung cancer tissue samples are immediately processed to ensure that the tissues retain most of their biological activities, providing guarantee for subsequent experiments;

[0048] (2) Pretreatment of tissue samples

[0049] The collected lung cancer tissue samples are washed with a pre-cooled 4°C tissue washing solution. When washing, shake quickly up, down, left and right with moderate shaking strength until the washing solution is clear. The tissue washing solution consists of the following components: 50 ml of phosphate buffered saline (PBS), 1x of Penicillin / Streptomycin, and 1x of Primocin antibiotic;

[0050] (3) Evaluation

[0051] Evaluate the content of epithelial cells in the obtained lung cancer tissue samples, and use ophthalmic scissors and forceps to remove non-epithelial components as much as possible, including muscle and adipose tissues, and remove obviously necrotic tissue components;

[0052] (4) Digestion of tissue blocks

[0053] At 37°C, a tissue digestion solution digestion and mechanical fragmentation method are selected to obtain cell clusters or single cells. The tissue digestion solution is guaranteed to be sterile, and the digestion time is controlled within 0.5 h and should not be too long, and it should be judged according to the size and degree of deterioration of the tissue blocks. The tissue digestion solution consists of the following components: 50 ml of Advance DMEM / F12 basal medium, 5 μM of ROCK inhibitor (ROCK inhibitor Y-27632), and 500 U / mL of type IV collagenase (IV Collagenase);

[0054] (5) Resuspension of cell precipitate and three-dimensional culture inoculation

[0055] Take an appropriate amount of the scaffold material Matrigel to resuspend the cell pellet. After mixing evenly, inoculate it into a culture well plate. Wait for the scaffold material to solidify, then add the lung cancer organoid medium to cover the cells, and place it in a 37°C carbon dioxide cell incubator for culture. The lung cancer organoid medium consists of the following components: 50 ml of Advance DMEM / F12 basal medium, 1x Glutamax, 1x HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1x B27 supplement, 1x Primocin antibiotic, 1x Penicillin / Streptomycin, 5 mM N-acetylcysteine, 5 μM ROCK inhibitor (ROCK inhibitor Y-27632), 200 ng / ml human Noggin, 0.2 μM R-Spondin1 (WNT signaling pathway activator), 200 ng / ml human epidermal growth factor (EGF), 5 mM Nicotinamide (Sirtuins inhibitor), 0.2 μM TGFβ inhibitor A83-01, 200 ng / ml fibroblast growth factor 10 (FGF10), 200 ng / ml recombinant human keratinocyte growth factor (FGF 7), 0.2 μM P38 MAPK inhibitor (SB202190).

[0056] (6) Organoid culture

[0057] According to the growth of the organoids, replace the lung cancer organoid medium once every 3 days.

[0058] In this example, identify the established organoids as needed, reasonably set the identification content and quality control of the organoids. The reference identification indicators are as follows: the morphology of the organoids under the microscope, the histological characteristics of the organoids, the cell viability of the organoids, the expression of common tumor markers in the organoids, etc.

[0059] Morphological detection of organoids under the microscope

[0060] On the second day of organoid culture, morphological observation can be carried out under an optical microscope. Lung cancer organoids usually form spherical structures with a diameter of 50 μm or more after several days of culture, and have good refractive properties. However, due to cancer heterogeneity differences, there may be significant individual differences in the size and morphology of tumor organoids. When conducting large-scale organoid culture and application, attention should be paid to the uniformity of the size, morphology, and quantity of organoids in each group. The histological characteristics of organoids, the cell viability of organoids, etc. are detected according to HE staining, immunohistochemistry, or immunofluorescence staining methods. Compare the expression of pathological markers in human-derived lung cancer organoids and lung cancer tissues, and detect the expression of proliferation indexes such as immunohistochemical Ki67 and TTF, etc. It can be seen that: from Figure 1 it can be clearly observed that the cultured tumor organoids have uniform morphology, large quantity, and high quality; from Figure 2 it can be clearly observed that the cultured tumor organoids have obvious vesicular characteristics of lung cancer organoids; from Figure 3 it can be clearly observed that the cultured tumor organoids have obvious vacuolar and grape-like characteristics of lung cancer organoids and are complete; from Figure 4 the morphology of tumor organoids in three-dimensional multicellular clusters can be clearly seen; from Figure 5 、 6 、7 and 8, the histological characteristics of lung cancer organoids can be clearly seen; Figure 9 This is the result diagram of immunohistofluorescence staining Ki67 detection of lung cancer organoids, and the expression of the proliferation index of the typical lung cancer marker Ki67 can be clearly seen; Figure 10 This is the result diagram of immunohistofluorescence staining TTF detection of lung cancer organoids, and the expression of the proliferation index of the typical lung cancer marker TTF can be clearly seen.

Claims

1. A method for culturing lung cancer tumor organoids, characterized in that, The following steps are included in sequence: (1) Selecting effective non-necrotic tissue from human lung cancer tissue as lung cancer tissue samples; (2) Cleaning the collected tissue samples; (3) evaluate tissue samples and remove non-epithelial components and tissue components with obvious necrosis; (4) selecting tissue digestion solution digestion and / or mechanical disruption methods to obtain cell clusters or single cells; (5) Cell pellet resuspension and three-dimensional culture inoculation Take the scaffold material and resuspend the cell pellet, mix well and then inoculate, or resuspend with lung cancer organoid culture medium and then inoculate, wait for the scaffold material to solidify, and add lung cancer organoid culture medium for culture; (6) Organoid culture.

2. The culture method of the lung cancer tumor organoid according to claim 1, wherein The following steps are included in sequence: (1) Obtaining tissue samples Selecting effective non-necrotic tissue from human lung cancer tissue obtained by puncture or surgery as lung cancer tissue samples, completely immersing the collected lung cancer tissue samples in sterile medical saline, storing them at a low temperature of 0°C to 4°C, and processing the lung cancer tissue samples in time within 0 to 8 hours; (2) Pretreatment of tissue samples The collected lung cancer tissue samples were washed with pre-cooled 4°C tissue washing solution until the washing solution became clear; (3) Evaluation of tissue samples To evaluate the epithelial cell content of lung cancer tissue samples obtained, remove non-epithelial components, and remove tissue components with obvious necrosis; (4) Digestion of tissue blocks Under the condition of 37°C, tissue digestion solution and / or mechanical disruption method are selected to obtain cell clusters or single cells; the tissue digestion solution is ensured to be sterile, and the digestion time is controlled within the time range of 0 to 2 hours; (5) Cell pellet resuspension and three-dimensional culture inoculation Take an appropriate amount of scaffold material to resuspend the cell pellet, mix well and inoculate it on the culture well plate, or resuspend it with lung cancer organoid culture medium and inoculate it on a 24-well plate. After the scaffold material solidifies, add lung cancer organoid culture medium to cover the cells and culture it in a 37°C carbon dioxide cell culture incubator; (6) Organoid culture Depending on the growth of the organoids, replace the lung cancer organoid culture medium every 3 to 5 days.

3. The culture method of the lung cancer tumor organoid according to claim 2, wherein: The tissue cleaning solution comprises the following components: 50ml of phosphate buffered saline, 1x penicillin-streptomycin and 1x primocin antibiotics.

4. The method for culturing lung cancer tumor organoids according to claim 1 or 2, characterized in that, The tissue digestion fluid comprises the following components: Advance DMEM / F12 basal medium 50 ml, ROCK inhibitor 2-10 μM and type IV collagenase 200-1000 U / mL or 2-5 mg / mL.

5. The method for culturing lung cancer tumor organoids according to claim 1 or 2, characterized in that: The scaffold material is matrix glue.

6. The culture method of the lung cancer tumor organoid according to claim 1 or 2, characterized in that, The lung cancer organoid culture medium comprises the following components: 50 ml of Advance DMEM / F12 basal medium, 1x glutamine, 1x 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1x B27 additive, 1x Primocin antibiotic, 1x penicillin-streptomycin double antibody, 2 - 5 mM of acetylcysteine, 2 - 10 μM of ROCK inhibitor, 50 - 250 ng / ml of human noggin, 0.1 - 2 μM of WNT signaling pathway activator, 50 - 250 ng / ml of human epidermal growth factor, 2 - 5 mM of Sirtuins inhibitor, 0.1 - 2 μM of TGFβ inhibitor A83-01, 50 - 250 ng / ml of fibroblast growth factor 10, 50 - 250 ng / ml of recombinant human keratinocyte growth factor, and 0.1 - 2 μM of P38 MAPK inhibitor.

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