Special culture medium added with S100 family recombinant protein for tumor organoids

Abstract

The invention belongs to the technical field of biological culture, and particularly relates to a special culture medium added with S100 family recombinant protein for tumor organoids. The invention relates to a special culture medium added with S100 family recombinant protein for tumor organs, which comprises a basic culture medium Advanced DMEM / F12, R-helix protein 1, head protein, a fibroblast growth factor 7, a fibroblast growth factor 10, a cell culture additive B27 supplement 50 *, a small-molecule inhibitor A83-01, a small-molecule inhibitor Y-27632, a small-molecule inhibitor SB202190, glutathione precursor N-acetylcysteine and nicotinamide. L-glutamine is used for replacing an additive GlutaMax 100 *, a pH buffer solution Hepes, an antibiotic and an antifungal agent-pririmosin, penicillin / streptomycin and S100 family recombinant protein. The special culture medium can better restore the real tumor microenvironment in vitro, enrich the culture system of the tumor organoid, and improve the culture success rate and growth rate of the tumor organoid.

Description

Technical Field

[0001] The invention belongs to the technical field of biological culture, and in particular relates to a special culture medium for tumor organoids added with S100 family recombinant proteins. Background Art

[0002] In recent years, the incidence and mortality of malignant tumors have continued to increase worldwide. In 2022, there will be approximately 20 million new cases of cancer worldwide. According to demographic forecasts, by 2050, the number of new cancer cases will reach 35 million. With the development of science and technology, various treatment methods for tumors (such as surgical treatment, radiotherapy, chemotherapy, molecular targeted drugs, immune checkpoint inhibitors, CAR-T cell therapy, cancer vaccines, endocrine and hormone therapy, radiofrequency ablation / microwave ablation, cryoablation, etc.) have emerged in an endless stream, but due to the large individual differences among tumor patients, ordinary treatment methods cannot solve the treatment problems of all patients. Therefore, seeking personalized medicine or precision medicine methods will be more conducive to improving patient survival rates.

[0003] Patient-derived organoids (PDOs) are three-dimensional miniature organ models formed by isolating cells from the patient's own tissue samples and culturing them in vitro. They retain the genomic characteristics, cell heterogeneity and physiological functions of the original tissue and are widely used in disease mechanism research, drug screening, personalized medicine and precision treatment strategy development. Patient-derived organoids are currently recognized as in vitro models that are closer to the actual situation of the patient's tumor, and can highly restore the molecular characteristics and pathological characteristics of the source tumor tissue in order to achieve the purpose of personalized medicine or precision medicine.

[0004] S100 protein (S-lflnprotei) plays a key role in regulating cell differentiation, proliferation, migration, apoptosis and other biological functions by interacting with various signal transduction proteins such as P53, β-catenins and nuclear factor-κB (NF-κB), thereby participating in tumorigenesis and tumor progression. At present, various tumor organoid culture systems are mainly based on the addition of basic cytokines. However, they have the following shortcomings: such as the inability to reproduce the real tumor microenvironment, ignoring the individualized needs caused by tumor heterogeneity, and the large differences in the success rate of tumor organoid culture. How to better improve the tumor organoid culture system, improve the success rate of tumor organoid culture, and realize the clinical application transformation of tumor organoids are currently difficult problems that need to be solved urgently. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a special culture medium for tumor organoids supplemented with S100 family recombinant proteins, which can better restore the real tumor microenvironment in vitro, enrich the culture system of tumor organoids, and improve the culture success rate and growth rate of tumor organoids.

[0006] In order to achieve the above objective, the following technical solution is adopted for a special culture medium for tumor organoids supplemented with S100 family recombinant proteins provided by this application:

[0007] A special culture medium for tumor organoids supplemented with S100 family recombinant proteins, comprising a basal medium Advanced DMEM / F12, R-spondin 1, noggin, fibroblast growth factor 7, fibroblast growth factor 10, cell culture additive B27 supplement 50×, small molecule inhibitor A83-01, small molecule inhibitor Y-27632, small molecule inhibitor SB202190, glutathione precursor N-acetylcysteine, nicotinamide, L-glutamine replacement additive GlutaMax 100×, pH buffer Hepes, antibiotic and antifungal agent - Primocin, penicillin / streptomycin, and S100 family recombinant proteins.

[0008] Preferably, the contents of each component are as follows: basal medium Advanced DMEM / F12, 1×; R-spondin 1, 250±10 ng / mL; noggin, 100±10 ng / mL; fibroblast growth factor 7, 25±5 ng / mL; fibroblast growth factor 10, 20±5 ng / mL; cell culture additive B27 supplement, 1×; small molecule inhibitor A83-01, 500±10 nM; small molecule inhibitor Y-27632, 5 μM; small molecule inhibitor SB202190, 500±10 nM; glutathione precursor N-acetylcysteine, 1.25±0.25 mM; nicotinamide, 10±2 mM; L-glutamine replacement additive GlutaMax, 1×; pH buffer Hepes, 10 mM; antibiotic and antifungal agent - Primocin, 50 μg / mL; penicillin / streptomycin, 100 mg / mL; S100 family recombinant proteins are all 20 ng / mL.

[0009] Another objective of the present invention is to provide a method for culturing tumor organoids supplemented with S100 family recombinant proteins.

[0010] In order to achieve the above objective, the following technical solution is adopted for a method for culturing tumor organoids supplemented with S100 family recombinant proteins provided by this application:

[0011] A method for culturing tumor organoids by adding recombinant proteins of the S100 family, comprising the following steps:

[0012] (1) Wash the tumor tissue with D-PBS buffer, cut the tumor tissue into small pieces, collect the small pieces into a centrifuge tube, add digestive solution, and then heat and digest the centrifuge tube.

[0013] (2) Add 10 mL of 1×Advanced DMEM / F12 containing 10 mM pH buffer Hepes, 1×L-glutamine substitute additive GlutaMax, 100 mg / mL penicillin / streptomycin, 5 μM Y-27632 to the above centrifuge tube to terminate digestion, centrifuge at room temperature, and discard the supernatant.

[0014] (3) Add 10 mL of red blood cell lysate to the cell pellet obtained by discarding the supernatant in step (2), resuspend the cells, add 10 mL of D-PBS buffer to terminate lysis after standing, centrifuge at room temperature, and discard the supernatant.

[0015] (4) Add 2 mL of the above-mentioned special medium for tumor organoids to the cell pellet in step (3), blow and resuspend the cells, centrifuge at room temperature, and discard the supernatant.

[0016] (5) Add 500 μL of the above-mentioned special medium for tumor organoids to the cell pellet in step (4), resuspend the cells and then perform cell counting; take 10,000 cells and mix them evenly according to the ratio of cell suspension: Matrigel = 1:1, drop the suspension to the center of a 24-well plate; place the 24-well plate in a water bath for a hot water bath, dry the bottom and invert it in a CO2 incubator, add 500 μL of the above-mentioned special medium for tumor organoids to each well, and culture at 37 °C and 5% CO2 concentration, change the medium every 3 days, and culture for 14 days to obtain tumor organoids.

[0017] Preferably, in step (1), the tumor tissue is cut into small pieces of 1 mm 3 , collect them into a 50 mL centrifuge tube, add 10 mL of digestive solution, and place the centrifuge tube in a 37 °C water bath for 1.5 h.

[0018] More preferably, the digestive solution includes 1×Advanced DMEM / F12 basal medium, 10 mM pH buffer Hepes, 1×L-glutamine substitute additive GlutaMax, 100 mg / mL penicillin / streptomycin, 5 μM Y-27632, 100 μg / mL type II collagenase, 1 μg / mL DNase I.

[0019] Preferably, in step (3), the centrifugation speed is 1400 rpm and the centrifugation time is 5 min.

[0020] Preferably, in step (4), the centrifugation speed is 1400 rpm and the centrifugation time is 5 min.

[0021] Preferably, in step (5), the temperature of the water bath is 37°C.

[0022] A third object of the present invention is to provide an application of a tumor organoid-specific medium supplemented with S100 family recombinant protein.

[0023] To achieve the above object, the application of a tumor organoid-specific medium supplemented with S100 family recombinant protein provided by the present application adopts the following technical solution:

[0024] An application of a tumor organoid-specific medium supplemented with S100 family recombinant protein, wherein the specific medium is used as a medium for the culture of breast cancer tissue, lung cancer tissue, gastrointestinal cancer tissue, head and neck cancer tissue, gynecological oncology tissue and metastatic tissues of the above tumors.

[0025] Beneficial effects:

[0026] The medium of the present invention is specifically supplemented with S100 family recombinant protein, which can better restore the real tumor microenvironment in vitro, enrich the culture system of tumor organoids, and improve the culture success rate and growth rate of tumor organoids.

[0027] The medium of the present invention can not only generally improve the culture success rate of tumor organoids, but also greatly improve the culture success rate in the culture of metastatic tumor organoids with strong tumor heterogeneity, and further improve the growth rate of metastatic tumor organoids. In addition, the tumor organoids formed by culturing with the medium of the present invention have a highly consistent gene typing with the patient's tumor tissue, providing a good beneficial choice for individualized medical guidance of tumor patients. Description of the drawings

[0028] The specification drawings forming a part of the present application are used to provide a further understanding of the present application. The schematic embodiments and descriptions thereof are used to explain the present application and do not constitute an improper limitation of the present application. Among them:

[0029] Figure 1 Bright field images of patient-derived breast cancer organoids cultured for 1 day and 14 days respectively using the medium and culture method in Example 2;

[0030] Figure 2 Bright field images of patient-derived left lung metastatic cancer organoids of rectal cancer cultured for the 1st day and the 14th day respectively using the medium and culture method in Example 3;

[0031] Figure 3Bright-field images of patient-derived breast cancer organoids cultured on the 1st and 14th days using the culture medium and culture method in Comparative Example 1;

[0032] Figure 4 Bright-field images of rectal cancer left lung metastatic cancer organoids formed by culturing on the 1st and 14th days using the culture medium and culture method in Comparative Example 2. Detailed implementation manners

[0033] The present application will be described in detail below with reference to embodiments. Each example is provided by way of explanation of the present application rather than limiting the present application. In fact, those skilled in the art will clearly understand that modifications and variations can be made to the present application without departing from the scope or spirit of the present application. For example, features shown or described as part of one embodiment can be used in another embodiment to yield yet another embodiment. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention shall fall within the scope of protection of the embodiments of the present invention.

[0034] Example 1

[0035] In this example, recombinant protein was prepared using S100A11 in the S100 family as an example.

[0036] The preparation method of the S100A11 recombinant protein in this example was referred to a preparation method of a nucleic acid, a recombinant plasmid, and a recombinant human IL-5 protein disclosed in Chinese Patent CN 118853713A.

[0037] 1) Construction of eukaryotic expression vector of S100A11 recombinant protein

[0038] The amino acid sequence of human S100A11 was obtained from the Uniprot database, and the gene sequence of human S100A11 was obtained from the NCBI database. A 6×His was connected to the C-terminus of human S100A11 for nickel column purification of the protein. The expression vector pCDNA3.1 was selected, and according to the characteristics of the S100A11 gene sequence, appropriate restriction enzyme sites were selected to ligate its gene sequence to the expression vector. Gene synthesis and sequencing were both completed by Sangon Biotech Co., Ltd.

[0039] 2) Transfection, expression, and harvesting of S100A11 recombinant plasmid

[0040] Cells in logarithmic growth phase were taken, and the corresponding transfection reagent was prepared to transfect the S100A11 plasmid into the cells. After 12 h of transfection, a new cell culture medium was replaced. The cell culture supernatant was collected at 36 h, 48 h, and 72 h after transfection and filtered using a 0.22 μm filter membrane.

[0041] 3) Purification of S100A11 recombinant protein

[0042] Using the sample purification method disclosed in Chinese invention patent application CN 118853713A, equilibrate the nickel column required for protein purification, pass the filtered cell culture supernatant through the column, purify the S100A11 recombinant protein, and detect the concentration of the S100A11 recombinant protein using an ultraviolet spectrophotometer.

[0043] The amino acid sequence of the S100A11 recombinant protein is as follows:

[0044] MAKISSPTETERCIESLIAVFQKYAGKDGYNYTLSKTEFLSFMNTELAAFTKNQKDPGVLDRMMKKLDTNSDGQLDFSEFLNLIGGLAMACHDSFLKAVPSQKRT.

[0045] Example 2

[0046] A special medium for tumor organoids supplemented with S100A11 recombinant protein. The content of each component of this medium is as follows: basal medium Advanced DMEM / F12, 1×; R-spondin 1, 250 ng / mL; Norrin, 100 ng / mL; fibroblast growth factor 7, 25 ng / mL; fibroblast growth factor 10, 20 ng / mL; cell culture additive B27 supplement, 1×; small molecule inhibitor A83-01, 500 nM; small molecule inhibitor Y-27632, 5 μM; small molecule inhibitor SB202190, 500 nM; glutathione precursor N-acetylcysteine, 1.25 mM; nicotinamide, 10 mM; L-glutamine replacement additive GlutaMax, 1×; pH buffer Hepes, 10 mM; antibiotic and antifungal agent - Primocin, 50 μg / mL; penicillin / streptomycin, 100 mg / mL; S100A11 recombinant protein, 20 ng / mL.

[0047] The culture method of the above-mentioned tumor organoids includes the following steps:

[0048] (1) Wash the surgically resected tumor tissue twice with D-PBS buffer, cut the tumor tissue into pieces of 1 mm 3 , collect them into a 50 mL centrifuge tube, add 10 mL of digestive solution. The digestive solution includes 1×Advanced DMEM / F12 basal medium, 10 mM pH buffer Hepes, 1×L-glutamine replacement additive GlutaMax, 100 mg / mL penicillin / streptomycin, 5 μM Y-27632, 100 μg / mL type II collagenase, 1 μg / mL DNase I. Place the centrifuge tube in a 37°C water bath and digest for 1.5 h.

[0049] (2) Add 10 mL of 1× Advanced DMEM / F12 containing 10 mM pH buffer Hepes, 1× L-glutamine substitute additive GlutaMax, 100 mg / mL penicillin / streptomycin, and 5 μM Y-27632 to terminate digestion, centrifuge at 1400 rpm for 5 min at room temperature, and discard the supernatant.

[0050] (3) Add 10 mL of red blood cell lysate to the cell pellet from which the supernatant was discarded in step (2), resuspend the cells, let stand for 10 min, then add 10 mL of D-PBS buffer to terminate lysis, centrifuge at 1400 rpm for 5 min at room temperature, and discard the supernatant.

[0051] (4) Add 2 mL of the above-mentioned special medium for tumor organoids to the cell pellet from which the supernatant was discarded in step (3), pipette to resuspend the cells, then centrifuge at 1400 rpm for 5 min at room temperature, and discard the supernatant.

[0052] (5) Add 500 μL of the above-mentioned special medium for tumor organoids to the cell pellet from which the supernatant was discarded in step (4), resuspend the cells and perform cell counting. Take 10,000 cells and mix them according to the ratio of cell suspension: Matrigel = 1:1, and pipette the suspension to the center of a 24-well plate. Place the 24-well plate in a 37°C water bath for 2 min, dry the bottom, invert it and place it in a CO2 incubator for 10 min, add 500 μL of the above-mentioned special medium for tumor organoids to each well, and culture at 37°C and 5% CO2 concentration. Change the medium every 3 days and culture for 14 days to obtain tumor organoids.

[0053] Figure 1 are bright-field images of patient-derived breast cancer organoids cultured using the above medium and culture method for 1 day and 14 days respectively. As Figure 1 can be seen, compared with the organoids cultured on the 1st day, the diameter of the breast cancer organoids cultured for 14 days is significantly increased and they are dense spheres with irregular edges, indicating that the above-mentioned special medium for tumor organoids can better support the growth of breast cancer organoids.

[0054] Example 3

[0055] A special medium for tumor organoids added with S100A11 recombinant protein and its culture method, using the special medium for tumor organoids and culture method in Example 2 to treat the left lung metastatic carcinoma tissue of rectal cancer in patients.

[0056] Figure 2 are bright-field images of patient-derived left lung metastatic carcinoma organoids of rectal cancer cultured on the 1st day and the 14th day. As Figure 2It can be seen that after culturing with the above-mentioned special medium for tumor organoids for 14 days, the organoids of the left lung metastatic carcinoma of rectal cancer derived from patients are dense and regular spheres, and about 85% of the organoids with a diameter of more than 50 μm account for the total number, fully indicating that the special medium for tumor organoids of the present invention can better support the culture of organoids of metastatic tumor tissues derived from patients.

[0057] Comparative Example 1

[0058] A method for culturing tumor organoids uses DMEN / F12 medium supplemented with 10% serum. The culturing process is the same as that in Example 2, and the tumor tissue is breast cancer tissue. Figure 3 Figure of bright field of breast cancer organoids formed by using the medium and culturing steps of this comparative example. Figure 3 It can be seen that breast cancer cells derived from patients cannot form breast cancer organoids under this system.

[0059] Comparative Example 2

[0060] A method for culturing organoids of metastatic tumors uses a medium without adding S100A11 recombinant protein. The culturing process of other tumor organoids and the used medium are the same as those in Example 2, and the tumor tissue is the left lung metastatic carcinoma tissue of rectal cancer.

[0061] Figure 4 Figure of bright field of organoids of the left lung metastatic carcinoma of rectal cancer formed by using the medium and culturing steps of this comparative example. Figure 4 It can be seen that after removing the S100A11 recombinant protein from the organoid medium, the diameter of the tumor organoids cultured for 14 days is significantly smaller, and about 90% of the organoids have a diameter of about 25 μm, with a slow growth rate.

[0062] The medium of the present invention specifically adds S100 family recombinant proteins. This medium can better restore the real tumor microenvironment in vitro, enrich the culturing system of tumor organoids, and improve the culturing success rate and growth rate of tumor organoids. This medium can not only generally improve the culturing success rate of tumor organoids, but also greatly improve the culturing success rate in the culturing of organoids of metastatic tumors with strong tumor heterogeneity, and further improve the growth rate of organoids of metastatic tumors. In addition, the gene typing of the tumor organoids formed by culturing with the medium of the present invention is highly consistent with that of the patient's tumor tissue, providing a very good and beneficial choice for individualized medical guidance for tumor patients.

[0063] In the present invention, the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0064] The foregoing are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A special medium for tumor organoids supplemented with recombinant proteins of the S100 family, characterized in that, It includes basal medium Advanced DMEM / F12, R-spondin 1, noggin, fibroblast growth factor 7, fibroblast growth factor 10, cell culture additive B27 supplement 50×, small molecule inhibitor A83-01, small molecule inhibitor Y-27632, small molecule inhibitor SB202190, glutathione precursor N-acetylcysteine, nicotinamide, L-glutamine replacement additive GlutaMax 100×, pH buffer Hepes, antibiotics and antifungal agent - Primocin, penicillin / streptomycin, and S100 family recombinant protein.

2. The tumor organoid-specific culture medium supplemented with the S100 family recombinant protein according to claim 1, wherein The content of each component is as follows: basal medium Advanced DMEM / F12, 1×; R-spondin 1, 250 ± 10 ng / mL; noggin, 100 ± 10 ng / mL; fibroblast growth factor 7, 25 ± 5 ng / mL; fibroblast growth factor 10, 20 ± 5 ng / mL; cell culture additive B27 supplement, 1×; small molecule inhibitor A83-01, 500 ± 10 nM; small molecule inhibitor Y-27632, 5 μM; small molecule inhibitor SB202190, 500 ± 10 nM; glutathione precursor N-acetylcysteine, 1.25 ± 0.25 mM; nicotinamide, 10 ± 2 mM; L-glutamine replacement additive GlutaMax, 1×; pH buffer Hepes, 10 mM; antibiotics and antifungal agent - Primocin, 50 μg / mL; penicillin / streptomycin, 100 mg / mL; S100 family recombinant protein is 20 ng / mL for all.

3. A method for culturing tumor organoids added with recombinant proteins of the S100 family as described in claim 1, characterized in that, It includes the following steps: (1) Wash the tumor tissue with D-PBS buffer, cut the tumor tissue into pieces, collect the pieces into a centrifuge tube, add digestive solution, and then heat and digest the centrifuge tube. (2) Add 10 mL of 1×Advanced DMEM / F12 containing 10 mM pH buffer Hepes, 1×L-glutamine replacement additive GlutaMax, 100 mg / mL penicillin / streptomycin, and 5 μM Y-27632 to the above centrifuge tube to terminate digestion, centrifuge at room temperature, and discard the supernatant. (3) Add 10 mL of red blood cell lysate to the cell pellet obtained by discarding the supernatant in step (2), resuspend the cells, add 10 mL of D-PBS buffer to terminate lysis after standing, centrifuge at room temperature, and discard the supernatant. (4) Add 2 mL of the above-mentioned special medium for tumor organoids to the cell pellet in step (3), pipette to resuspend the cells, centrifuge at room temperature, and discard the supernatant. (5) Add 500 μL of the above-mentioned special medium for tumor organoids to the cell pellet in step (4), resuspend the cells and perform cell counting; take 10,000 cells and mix them evenly according to the ratio of cell suspension: Matrigel = 1:1, and place the suspension in the center of a 24-well plate; place the 24-well plate in a water bath for a hot water bath, dry the bottom and invert it in a CO2 incubator, add 500 μL of the above-mentioned special medium for tumor organoids to each well, and culture at 37 °C and 5% CO2 concentration, change the medium every 3 days, and culture for 14 days to obtain tumor organoids.

4. The culture method of tumor organoids adding S100 family recombinant protein according to claim 3, characterized in that, In step (1), the tumor tissue is cut into fragments of 1 mm 3 , collected in a 50 mL centrifuge tube, 10 mL of digestive solution is added, and the centrifuge tube is placed in a water bath at 37 °C for digestion for 1.5 h.

5. The method for culturing tumor organoids adding S100 family recombinant protein according to claim 4, characterized in that, The digestive solution includes 1×Advanced DMEM / F12 basal medium, 10 mM pH buffer Hepes, 1×L-glutamine substitute additive GlutaMax, 100 mg / mL penicillin / streptomycin, 5 μM Y-27632, 100 μg / mL type II collagenase, 1 μg / mL DNase I.

6. The culture method of tumor organoids adding S100 family recombinant protein according to claim 3, characterized in that, In step (3), the centrifugation speed is 1400 rpm and the centrifugation time is 5 min.

7. The method for culturing tumor organoids by adding S100 family recombinant protein according to claim 3, wherein In step (4), the centrifugation speed is 1400 rpm and the centrifugation time is 5 min.

8. The culture method of tumor organoids adding S100 family recombinant protein according to claim 3, characterized in that, In step (5), the temperature of the water bath is 37 °C.

9. Use of a tumor organoid-specific culture medium supplemented with a recombinant protein of the S100 family as described in claim 1, characterized in that, The special medium is used as a medium for the culture of breast cancer tissue, lung cancer tissue, gastrointestinal cancer tissue, head and neck cancer tissue, gynecological tumor tissue and the above-mentioned tumor metastatic tissues.

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