Preparation method of tumor organoid

Abstract

The invention belongs to culture of tumor organoids, and particularly relates to a preparation method of tumor organoids, which comprises the following steps: digesting a lung tumor sample to obtain an epithelium starting material, performing three-dimensional culture on a basement membrane matrix to form primary organoids, and performing early cryopreservation in a main library; homologous organs are homogenized and subpackaged into test groups, and after adaptive culture is carried out under a combination of multiple culture media, chemotherapy pressure is applied, and elution recovery is carried out; a target culture medium combination for screening the drug-resistant subgroups is determined according to survival and regrowth and in combination with a normal sample removal result of morphology; according to the method, the proportion of tumor components is increased, the drug-resistant survival subgroups are enriched, and the preparation consistency and repeatability are improved.

Description

TECHNICAL FIELD

[0001] The application belongs to tumor organoid culture, and particularly relates to a tumor organoid preparation method. BACKGROUND

[0002] The individualized drug evaluation and mechanism research of tumors require a culture model which can maintain the growth of tumor epithelial cells in vitro for a long time and reflect the tumor characteristics of patients as much as possible.

[0003] Patient-derived organoids (which can be referred to as tumor organoids for short) are usually formed into self-organized epithelial structures by adopting a three-dimensional basement membrane matrix embedding culture method, and can be used for tumor biological research and in-vitro drug screening, and are used to construct a tumor organoid library to support multi-drug comparison and repeated verification.

[0004] In the process of establishing tumor organoids, the starting sample often contains normal tissue epithelial components and tumor epithelial components. In order to improve the proportion of tumor components and inhibit the competitive expansion of normal-derived organoids, the existing technology usually sets selection pressure on the culture conditions, and one common strategy is to omit exogenous Wnt ligands / factors in the tumor organoid culture medium, such as not adding Wnt3A conditioned medium or Wnt substitutes, for promoting the selective outgrowth of tumor cell populations. However, the pressure from the culture conditions can increase the purity of the tumor, but at the same time, it can also reduce the heterogeneity of the tumor, that is, the clones / subpopulations more suitable for the culture conditions are preferentially retained and expanded, and the subpopulations sensitive or dependent on different ecological niche factors are suppressed. Taking lung cancer as an example, small cell lung cancer (SCLC) accounts for 20% of lung cancer, and non-small cell lung cancer (NSCLC) accounts for 80%, including adenocarcinoma (AD), squamous cell carcinoma (SCC), and large cell carcinoma. The genetic heterogeneity within the tumor affects the tumor immune microenvironment, which not only increases the risk of lung tumor metastasis, but also is an important reason for drug resistance in lung cancer treatment.

[0005] For example, the growth of some tumor subpopulations still depends on the supply of exogenous Wnt signal ligands or the enhancement of Wnt signals by R-spondin. When the culture medium adopts conditions such as removing Wnt3A conditioned medium or Wnt substitutes, such tumor subpopulations may be inhibited or even eliminated together with normal intestinal epithelial cells, thereby further weakening the coverage of the culture to the tumor internal heterogeneity subpopulation.

[0006] This leads to the tumor treatment drugs screened only show sensitivity to part of the clones or subpopulations selected and expanded under the culture condition pressure, and it is difficult to cover the differences of different subclones in the tumor and the potential resistance risk, thereby deviating the prediction of the in vitro drug screening conclusion to the overall efficacy of the patient. SUMMARY

[0007] The present application aims to provide a preparation method of tumor organoids to solve the above problems, comprising the following steps: S1, obtaining a tumor tissue sample, mechanically shearing and enzyme digestion, filtering through a screen and washing by centrifugation to obtain an epithelial cell mass, gland-like structure or cell suspension; S2, mixing and embedding the starting material with a basement membrane matrix, adding an expansion culture medium for three-dimensional culture after gelation, and obtaining a primary organoid; S3, recovering part of the organoids as a master batch and freezing after the primary organoids reach a preset growth stage; S4, recovering the unfrozen part and slightly dividing, and uniformly packaging into several test organoid groups; S5, configuring a basic medium BM, and forming a plurality of medium factor combinations based on the basic medium BM, so that each test group is adapted to culture under different combination conditions; S6, applying consistent chemotherapy drug treatment conditions to each test group; S7, removing the drug-containing medium and eluting, and recovering and culturing for a preset time window under the corresponding medium factor combination conditions; S8, taking the survival after chemotherapy and the recovery of the regrowth ability during the recovery period as the basis for determination and morphological confirmation, eliminating the culture results dominated by normal sample organoids, and counting the surviving tumor subpopulations; S9, determining the target medium factor combination according to the determination results of the surviving tumor subpopulations; S10, thawing and recovering the master batch, and uniformly expanding and passing in the target medium factor combination to obtain a tumor organoid culture.

[0008] Further, the tumor tissue sample is a lung cancer tissue sample, preferably a fresh tissue obtained and stored under sterile conditions.

[0009] Further, the basic medium uses Advanced DMEM / F12 as the base liquid and adds GlutaMax, HEPES, double antibodies, N2 supplement and B27 supplement.

[0010] Further, the medium factor combination is not less than two.

[0011] Further, the chemotherapy drug is not less than two.

[0012] Further, the culture medium factor combination comprises: Combination A: Wnt3a + R-spondin 1 + Noggin + EGF; Combination B: R-spondin 1 + Noggin + EGF; Combination C: EGF, no R-spondin 1 or low dose supply.

[0013] Further, the tumor tissue is lung cancer tissue, the three chemotherapy drugs in S5 include cisplatin.

[0014] Further, the S1 also acquires adjacent relatively normal lung tissue or airway epithelial-derived samples, and is prepared and cultured to form a normal lung epithelial organoid control in the manner of S1-S2.

[0015] Further, the second chemotherapy drug in S5 includes any one of the following: pemetrexed, gemcitabine, paclitaxel, docetaxel, vinorelbine, and etoposide.

[0016] Further, in S5, cisplatin is applied first, and then cisplatin and the second chemotherapy drug are applied.

[0017] The beneficial effects achieved by the present application with the above structure are as follows: In early establishment of a recoverable master library batch and cryopreservation, different culture pressure conditions are applied to each test group first, and then consistent chemotherapy treatment and elution recovery processes are applied to determine the tumor subpopulation with the greatest tumor drug resistance, and the master library is subsequently recovered and expanded under the culture medium combination selected for this subpopulation, which can effectively screen drug-resistant tumor subpopulations, so as to facilitate the use of tumor organoids to screen other drugs for use in combination. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The tumor organoid culture medium combination and expansion process diagram of the present application is determined. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0020] The present application does not initially preset a unique culture medium, but after the establishment of the primary organoids, the homologous organoids are divided into multiple test groups and placed in different combinations of culture medium factors for adaptive culture; after adaptation, the same chemotherapy drug pressure treatment is applied to each group, and then elution and recovery culture is carried out, and the performance of "still surviving after chemotherapy and recovering the growth ability in the recovery period" is taken as the main selection index, so as to determine the most suitable culture medium factor combination X for obtaining the target surviving subpopulation; finally, the combination X is used for unified recovery and expansion of the frozen main library batch to obtain tumor organoids.

[0021] As shown in the figure, the present application proposes a preparation method of tumor organoids, comprising the following steps: S1, tissue acquisition and starting material preparation; Obtain a tumor tissue sample, which can be a surgical resection tissue; or a biopsy tissue; preferably a fresh tissue obtained under sterile storage conditions.

[0022] After the tissue is mechanically cut and shredded, enzyme digestion is carried out, and then filtered through a screen and washed by centrifugation to enrich epithelial cell clumps, gland-like structures or cell suspensions, to obtain epithelial-derived starting materials that can be used for three-dimensional embedding culture.

[0023] S2, three-dimensional embedding and primary culture; Mix the epithelial-derived starting material with the basement membrane matrix and embed it by dispensing, and after gelation, add the expansion culture medium for three-dimensional culture to obtain the primary organoids.

[0024] S3, main library batch freezing; After the primary organoids are formed and reach the preset growth stage, one part of the organoids is recovered as the main library batch and frozen for storage, which is used for subsequent unified recovery and expansion.

[0025] S4, test group packaging; The organoids that do not enter the main library freezing are recovered and slightly segmented, and are uniformly packaged into several test organoid groups.

[0026] S5, culture with the configuration of the basic culture medium BM and different culture medium factor combinations; The basic culture medium BM is configured, and various culture medium factor combinations are formed based on BM, so that each test organoid group is adaptively cultured under different combination conditions.

[0027] S6, chemotherapy drug pressure screening treatment; According to the target cancer species, the chemotherapy drugs or chemotherapy drug combinations used for in vitro pressure screening are determined in advance, and consistent chemotherapy treatment conditions are applied to each test organoid group, so that each group receives drug pressure under the same drug composition, concentration window and treatment time.

[0028] S7, elution and recovery culture; After the chemotherapy drug screening process is completed, the drug-containing medium is removed and eluted, and the pre-set time window is continued to recover culture under the respective corresponding medium factor combination conditions.

[0029] S8, survival subpopulation determination and morphological confirmation; Survival after chemotherapy and maintaining / restoring regrowth ability during the recovery period are used as the main determination basis, and morphological confirmation is performed. The culture results with normal organoids are excluded, and the survival tumor subpopulation is counted.

[0030] S9, determine the target culture medium factor combination; Based on the survival tumor subpopulation determination result of S8, the target culture medium factor combination X is determined from the combination as the culture condition for subsequent unified expansion.

[0031] S10, main library recovery and unified expansion After the main library batch is thawed and recovered, it is uniformly expanded in the target culture medium factor combination X to obtain tumor organoid culture, and can be further frozen to form a reusable library.

[0032] Example 1, the pre-determined chemotherapy drug is cisplatin, and the purpose is to screen another drug for use. This example is used to obtain a lung tumor subpopulation that can still survive and restore regrowth ability under cisplatin pressure, so as to use the finally cultured lung cancer organoids to screen another drug. In this example, lung cancer tissue samples are first obtained, which can be surgical resection tissue or biopsy tissue; preferably fresh and under sterile storage conditions.

[0033] The tissue is mechanically cut and then enzymatically digested, and then filtered through a screen and centrifuged and washed to enrich epithelial cell clumps, gland-like structures or cell suspensions, to obtain epithelial-derived starting material that can be used for three-dimensional embedding culture.

[0034] According to the above general steps S1-S4, the primary organoids are established and the main library batch is prepared, and a plurality of test organoid groups are obtained.

[0035] The base medium BM is configured, and a plurality of medium factor combinations are formed based on the BM, so that each test organoid group is adapted to culture under different combination conditions.

[0036] The base medium BM uses Advanced DMEM / F12 as the base liquid and adds GlutaMax, HEPES, double antibodies, N2 supplement and B27 supplement; the medium factor combination is used to form different culture condition pressures.

[0037] The medium factor combination can include at least the following three combinations for parallel control and adaptation culture: Combination A: BM + Wnt3a + R-spondin 1 + Noggin + EGF; Combination B: BM + R-spondin 1 + Noggin + EGF, without Wnt3a; Combination C: BM + EGF, with R-spondin 1 and / or Noggin withdrawn or supplied at low dose.

[0038] The above combinations are used to form differentiation pressure at the level of culture conditions, so that different cell populations dependent on niche factors can present a comparable adaptive state before subsequent cisplatin screening.

[0039] The test groups under combinations A-C are subjected to uniform chemotherapy pressure according to the method of S6: The chemotherapy drug used for in vitro pressure screening is cisplatin according to the target cancer, and uniform cisplatin treatment conditions are applied to each test organoid group, so that each group receives drug pressure under the same drug composition, concentration window and treatment duration to obtain comparable screening results.

[0040] After completing the cisplatin screening treatment, the drug-containing culture medium is removed and eluted, and the pre-set time window is continued under the corresponding medium factor combination conditions.

[0041] Survival after cisplatin treatment and maintenance / restoration of regrowth ability during the recovery period are used as the main basis for determination, and morphological confirmation is performed to exclude culture results dominated by normal sample organoids, and the culture medium of the surviving tumor subpopulation is used as the target combination X.

[0042] The target combination X is determined from combinations A-C, the main library batch is thawed, and combination X is used for amplification and passage to obtain tumor organoid cultures and store them.

[0043] If all test organoid groups do not recover under cisplatin pressure, the culture medium combination can be further changed.

[0044] Example 2, under the premise that cisplatin is the main drug, different second drugs are used to apply chemotherapy pressure, and the tumor organoids prepared are used to screen the third drug.

[0045] First, the culture of lung cancer organoids is performed according to S1-S5, tumor tissue samples are obtained and epithelial-derived starting materials are prepared; three-dimensional embedding forms primary organoids; early establishment of main library batch and storage; the non-frozen part is uniformly divided into several test organoid groups; the basic culture medium BM is configured and various medium factor combinations are formed, so that each test group is adapted to culture under different combination conditions; Among them, preferably, in addition to obtaining lung cancer tumor tissue samples, adjacent relatively normal lung tissue or airway epithelial-derived samples can also be obtained under the same patient conditions, and normal lung epithelial organoid controls are prepared and cultured in the manner of S1-S2.

[0046] According to the target cancer, cisplatin-based chemotherapy drugs are predetermined, and candidate chemotherapy drug combinations are set. Each test organoid group is subjected to chemotherapy drug pressure treatment. The candidate combination library can include: D1: Cisplatin single drug; D2: Cisplatin + pemetrexed; D3: Cisplatin + gemcitabine; D4: Cisplatin + paclitaxel; D5: Cisplatin + docetaxel; D6: Cisplatin + vinorelbine; D7: Cisplatin + etoposide.

[0047] Preferably, the same concentration gradient window is used for all candidate combinations, and the treatment time is kept consistent between different medium factor combinations, only the composition of the second drug is changed to ensure comparability.

[0048] After completing the chemotherapy drug pressure treatment, the drug-containing culture medium is removed and eluted, and the corresponding medium factor combination is continued for a predetermined time window.

[0049] In this embodiment, not only the surviving tumor subpopulation is counted, but also the normal lung epithelial organoid culture results are counted. For the test group tumor organoids, the survival after chemotherapy and the ability to maintain / regain growth during the recovery period are used as the main determination basis, and morphological confirmation is performed to exclude culture results dominated by normal samples, and the surviving tumor subpopulation is counted.

[0050] For the normal lung epithelial organoid control, the regrowth performance during the recovery period is used as the toxicity evaluation index, and the index includes: the recovery of the number of organoids, the increase in diameter, the increase in volume, and the structural integrity. Preferably, the normal lung epithelial organoid also superimposes ATP activity as a cell activity indicator.

[0051] Based on the above determination results, the chemotherapy drug combination that minimally affects the number recovery, diameter or volume increase, and structural integrity of the normal lung epithelial organoid control under the same concentration window and treatment duration is determined; And under the condition of the target chemotherapy drug combination, the tumor subpopulation that still survives and can regrow, and the morphological confirmation shows that the tumor sample organoid is dominant in the culture medium factor combination.

[0052] After thawing and recovery of the master library batch, the tumor organoid cultures screened under the condition of the target chemotherapy drug combination are obtained by uniformly expanding and passing in the target medium factor combination.

[0053] Example 3 is a combined implementation of Example 1 and Example 2, first according to Example 1, in S5, the test group tumor organoids are subjected to in vitro drug pressure screening with cisplatin, and after elution and recovery, tumor subpopulation organoids that survive cisplatin pressure and can be regrown are obtained; Then according to Example 2, the cisplatin pressure surviving tumor subpopulation organoids are used as test materials in Example 2, and candidate second drugs are compared in parallel under the condition that cisplatin is the main drug, and the minimum toxicity combination of cisplatin + second drug is determined based on the degree of inhibition of regrowth during the recovery period; Subsequently, the master library is uniformly recovered and expanded under the medium factor combination corresponding to the minimum toxicity combination, and the obtained tumor organoids are further used to determine a third drug.

[0054] The above only describes specific embodiments of the present application and is not used to limit the present application. For those skilled in the art, various modifications, equivalent replacements or improvements to the above embodiments without departing from the technical concept of the present application shall fall within the protection scope of the present application. The protection scope of the present application is limited by the scope defined in the claims, and the specification and drawings can be used to explain the claims.

Claims

1. A method for preparing tumor organoids, characterized in that, Includes the following steps: S1. Obtain tumor tissue samples, mechanically cut and enzymatically digest them, filter them through a sieve and wash them by centrifugation to obtain epithelial cell clumps, gland-like structures or cell suspensions; S2. Mix the starting material with the basement membrane matrix, apply glue and embed, gel, add amplification medium and culture in three dimensions to obtain the first generation of organoids. S3. After the first-generation organoids reach the preset growth stage, a portion of the organoids are recovered as the master library batch and cryopreserved. S4. The unfrozen portion was recovered and lightly divided, then homogenized and packaged into several test organoid groups; S5. Prepare the basic culture medium BM, and on the basis of the basic culture medium BM, form a variety of culture medium factor combinations so that each test group can be adapted to culture under different combination conditions. S6. Apply the same chemotherapy drug treatment to all test groups; S7. Remove the drug-containing culture medium and wash it off. Under the corresponding combination of culture medium factors, revive and culture for a preset time window. S8. Based on the survival of patients after chemotherapy and their ability to regenerate during the recovery period, and morphological confirmation, culture results with dominant normal organoids were excluded, and surviving tumor subpopulations were statistically analyzed. S9. Determine the target culture medium factor combination based on the results of the surviving tumor subpopulation determination; S10. After thawing and resuscitating the main batches, amplify and passage them uniformly in the target culture medium factor combination to obtain tumor organoid cultures.

2. The method for preparing tumor organoids according to claim 1, characterized in that, The tumor tissue sample is a lung cancer tissue sample, preferably a freshly obtained tissue that is under sterile preservation conditions.

3. The method for preparing tumor organoids according to claim 1, characterized in that, The basal culture medium used Advanced DMEM / F12 as the base medium and supplemented with GlutaMax, HEPES, antibiotics, N2 supplement and B27 supplement.

4. The method for preparing tumor organoids according to claim 1, characterized in that, The culture medium factor combination shall be no less than two.

5. The method for preparing tumor organoids according to claim 1, characterized in that, The chemotherapy drugs are no less than two.

6. The method for preparing tumor organoids according to claim 4, characterized in that, The culture medium factor combination includes: Combination A: Wnt3a + R-spondin 1 + Noggin + EGF; Combination B: R-spondin 1 + Noggin + EGF; Combination C: EGF, without R-spondin 1 or at low doses.

7. The method for preparing tumor organoids according to claim 5, characterized in that, The tumor tissue is lung cancer tissue, and there are three types of chemotherapy drugs in S5, including cisplatin.

8. The method for preparing tumor organoids according to claim 7, characterized in that, In step S1, samples of adjacent relatively normal lung tissue or airway epithelium are also obtained and prepared and cultured in the manner of S1–S2 to form normal lung epithelial organoid controls.

9. The method for preparing tumor organoids according to claim 8, characterized in that, The second chemotherapy drug in S5 includes any one of the following: pemetrexed, gemcitabine, paclitaxel, docetaxel, vinorelbine, or etoposide.

10. A method for preparing a tumor organoid according to any one of claims 7-9, characterized in that, In S5, cisplatin was administered first, followed by cisplatin and the second chemotherapy drug.

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