Pancreatic cancer organoid culture medium and pancreatic cancer organoid culture method

Abstract

The invention provides a pancreatic cancer organoid culture medium and a pancreatic cancer organoid culture method, and belongs to the technical field of organoid culture. The pancreatic cancer organoid culture medium is obtained by combining specific types of basic culture media, additives, cell factors and inhibitors, and the problems that in the prior art, in pancreatic cancer organoid culture, the product size is small, and the activity needs to be improved can be solved. When the culture medium provided by the invention is used for culturing the pancreatic cancer organoid, the size and activity of the obtained organoid can be remarkably improved, a higher cell proliferation speed can be obtained in combination with the culture method provided by the invention, and efficient production of the pancreatic cancer organoid is expected to be realized when the culture medium is applied to the industry. A better material is provided for in-vitro tumor biology and related researches on treatment, and the material has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to a pancreatic cancer organoid culture medium and a pancreatic cancer organoid culture method. Background Art

[0002] Tumor organoids are three-dimensional models that are highly similar to the patient's tumor characteristics, which are established by using fresh tumor tissue from patients through an in vitro three-dimensional (3D) cell culture system. Therefore, "organoids" means similar to tissue organs. They can serve as a substitute for patients and become an in vitro model for clinical guidance of personalized drug use.

[0003] In 2009, scholars in this field used adult stem cells from the mouse intestine to cultivate the first miniature intestinal organoid, which set off a wave of organoid research. In 2011, researchers used organoid technology to first establish tumor organoids. They cultivated human small intestine and colon organoids by adjusting the culture environment based on the culture conditions of mouse colon crypts, and then further optimized and adjusted them to establish tumor organoid models from colon adenomas and adenocarcinomas and Barrett's esophagus.

[0004] The establishment of tumor organoid models derived from cancer patients is considered to be another major breakthrough in organoid culture technology. Tumor organoids are cell clusters formed by three-dimensional tissue culture of tumor-specific stem cells in tumor tissues. They can simulate the characteristics of tumors in vivo and the heterogeneity of tumor cells. The establishment of this technology has not only provided a reliable model for cancer research and treatment, but also opened up new horizons for personalized cancer treatment. At present, researchers have successfully produced many tumor organoids by optimizing various culture conditions, including colon cancer, prostate cancer, gastric cancer, breast cancer and pancreatic cancer, endometrial / ovarian cancer, uterine carcinosarcoma, urothelial carcinoma and renal cancer. Compared with traditional tumor research models, tumor organoids can simulate the structure and function of tumors in vivo to the greatest extent, can intuitively show the process of tumor growth, and can reflect individual differences among patients. It is an intuitive, reliable, efficient and ethically controversial organ-level research system, which is more suitable for in vitro tumor biology and treatment-related research.

[0005] Chinese patent CN116555181A discloses a pancreatic cancer organoid, culture medium and culture method. The pancreatic cancer organoid culture medium is characterized by including basic culture and specific added factors. The pancreatic cancer organoid culture medium provided by the invention contains the minimum components required for pancreatic cancer organoid culture, and the components do not require bovine serum albumin, the most common component in cell culture, which saves costs while reducing the cytotoxicity and inhibitors brought by FBS; the pancreatic cancer organoid culture medium provided by the invention is suitable for the culture of pancreatic cancer organoids, and the various components of the organoid culture medium produce a synergistic effect, so that the pancreatic cancer organoids obtained by culture maintain the morphological structure and genetic characteristics of the primary tissue.

[0006] However, the above culture method can only obtain pancreatic cancer organoids with a size of about 200 μm, and the activity needs to be improved. Summary of the invention

[0007] The object of the present invention is to provide a pancreatic cancer organoid culture medium and a pancreatic cancer organoid culture method, which can obtain pancreatic cancer organoids of larger size and with better activity.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a pancreatic cancer organoid culture medium, comprising the following components:

[0010] basal media, supplements, cytokines, and inhibitors;

[0011] The cytokines include R-Spondin 1, Noggin, epidermal growth factor and B27supplement.

[0012] Preferably, in the pancreatic cancer organoid culture medium, the final concentration of R-Spondin 1 is 50 to 150 ng / ml;

[0013] The final concentration of Noggin is 50-150 ng / ml;

[0014] The final concentration of the epidermal growth factor is 50-150 ng / ml;

[0015] The B27 supplement is used at a dilution concentration of 1X.

[0016] Preferably, the cytokines further include fibroblast growth factor 10;

[0017] Preferably, the final concentration of the fibroblast growth factor 10 is 30-80 ng / ml.

[0018] Preferably, the inhibitors include N-acetylcysteine, nicotinamide, A83-01 and Y-27632;

[0019] Preferably, in the pancreatic cancer organoid culture medium, the final concentration of N-acetylcysteine ​​is 0.5 to 2 mM;

[0020] The final concentration of nicotinamide is 5-20 mM;

[0021] The final concentration of A83-01 is 0.3-0.8 μM;

[0022] The final concentration of Y-27632 is 5-20 μM.

[0023] Preferably, the inhibitor further comprises gastrin;

[0024] Preferably, in the pancreatic cancer organoid culture medium, the final concentration of gastrin is 5 to 20 nM.

[0025] Preferably, the additive comprises one or more of 4-hydroxyethylpiperazineethanesulfonic acid, antibiotics and L-alanyl-L-glutamine;

[0026] Preferably, in the pancreatic cancer organoid culture medium, the final concentration of the 4-hydroxyethylpiperazineethanesulfonic acid is 5 to 20 mM;

[0027] The final concentration of the antibiotic is 1X;

[0028] The final concentration of L-alanyl-L-glutamine was 2 mM.

[0029] Preferably, the basal culture medium is Advanced DMEM / F12.

[0030] The present invention also provides a pancreatic cancer organoid culture method, comprising the following steps:

[0031] The pancreatic cancer tissue is mixed with a digestive fluid and subjected to a digestion process to obtain a digested tissue;

[0032] The digested tissue was filtered through a cell strainer to obtain a precipitate;

[0033] The precipitate is purified to obtain cells;

[0034] The cells are inoculated into the above pancreatic cancer organoid culture medium for organoid growth culture to obtain pancreatic cancer organoids.

[0035] Preferably, the digestive fluid contains type 2 collagenase and neutral protease.

[0036] Preferably, the final concentration of type 2 collagenase in the digestive solution is 1 to 3 mg / ml;

[0037] The final concentration of the neutral protease in the digestive solution is 0.01-0.03 U / ml;

[0038] And / or, the digestion treatment time is 1 to 2 hours.

[0039] Beneficial effects of the present invention:

[0040] The present invention obtains a pancreatic cancer organoid culture medium by combining specific types of basal culture medium, additives, cytokines and inhibitors. The culture medium can significantly improve the size and activity of the obtained organoids by culturing pancreatic cancer organoids. In combination with the culture method provided by the present invention, a faster cell proliferation rate can be obtained. The industry is expected to achieve efficient production of pancreatic cancer organoids, provide better materials for in vitro tumor biology and treatment-related research, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Figure 1 shows the culture conditions on the first and third days after cell inoculation in Example 1;

[0042] Figure 2 Figure 2 shows the culture conditions on the first and third days after cell inoculation in Example 2;

[0043] Figure 3 This is a diagram of the pancreatic cancer organoids finally obtained in Example 1. DETAILED DESCRIPTION

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] Prepare the culture medium:

[0047] Table 1 Example 1 Culture medium formula

[0048]

[0049] To cultivate:

[0050] Fresh pancreatic cancer tissue samples were collected and immediately placed in tissue protection solution and stored at 4°C.

[0051] Add ice-cold PBS containing 1% double antibody to a 35 mm dish, remove the pancreatic cancer tissue sample, remove normal tissue, fat, blood clots and necrotic parts, and cut the pancreatic cancer sample into 1-2 mm pieces with surgical scissors or a scalpel. 3 Small fragments.

[0052] The tissue fragments were transferred into a 10 ml centrifuge tube, and the tissue was allowed to settle under gravity. The supernatant was removed and washed twice with ice-cold PBS containing 1% double antibody.

[0053] Add 5 ml of pancreatic cancer organoid digestion solution (2 mg / ml type 2 collagenase, 1 U neutral protease) according to the amount of tissue, seal with sealing film, and digest on a shaker at 37°C for 1.5 hours, shaking and mixing on a shaker every 30 minutes.

[0054] After digestion is complete, pipette up and down 10 times with a 1 ml pipette tip to further digest the tissue, filter it using a 100 μm cell strainer, centrifuge at 300 g for 5 min, and remove the supernatant.

[0055] Add 5 ml adDMEM / F12+++ (containing double antibody, glutamine, HEPES) and resuspend, centrifuge at 300g for 5 minutes, and remove the supernatant.

[0056] According to the amount of precipitation, add the corresponding volume of pancreatic cancer organoid culture medium and matrix gel in a 1:1 ratio to resuspend.

[0057] Take out the 24-well plate placed in the 37°C incubator 2 hours in advance, add 50 μl of the mixed solution to each well, put it in the 37°C incubator for 5 minutes, and then invert it to solidify for 20 minutes.

[0058] After solidification, add 500 μl of pancreatic cancer organoid culture medium to a 24-well plate and place it in a cell culture incubator for culture. Observe the growth of the organoids every day and replace the culture medium every 2 to 3 days.

[0059] Cultivate photos such as Figure 1 As shown, the survival rate on the first day was 90%, the average size of the organoids on the third day was 250 μm, and the final product was obtained on the seventh day, with an average organoid size of 600 μm. Figure 3 shown.

[0060] Example 2

[0061] Table 2 Example 2 Culture medium formula

[0062]

[0063]

[0064] The culture scheme is the same as in Example 1.

[0065] Cultivate photos such as Figure 2 As shown, the survival rate on the first day was 50%, the average size of the organoids on the third day was 200 μm, and the final product was obtained on the 7th day with an average size of 300 μm.

[0066] The results showed that the organoids grew very well on the first day in Example 1 provided by the present invention, while the organoids in Example 2 showed apoptosis on the first day; the organoids in Example 1 grew faster and larger on the first day, while the organoids in Example 2 grew slower, but the final cultured size was still better than the organoids in the prior art (about 100 μm).

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pancreatic cancer organoid culture medium, characterized in that Includes the following components: basal media, supplements, cytokines, and inhibitors; The cytokines include R-Spondin 1, Noggin, epidermal growth factor and B27supplement.

2. The pancreatic cancer organoid culture medium according to claim 1, characterized in that In the pancreatic cancer organoid culture medium, the final concentration of R-Spondin 1 is 50-150 ng / ml; The final concentration of Noggin is 50-150 ng / ml; The final concentration of the epidermal growth factor is 50-150 ng / ml; The B27 supplement is used at a dilution concentration of 1X.

3. The pancreatic cancer organoid culture medium according to claim 1, characterized in that The cytokines also include fibroblast growth factor 10; Preferably, the final concentration of the fibroblast growth factor 10 is 30-80 ng / ml.

4. The pancreatic cancer organoid culture medium according to claim 3, characterized in that The inhibitors include N-acetylcysteine, nicotinamide, A83-01, and Y-27632; Preferably, in the pancreatic cancer organoid culture medium, the final concentration of N-acetylcysteine ​​is 0.5 to 2 mM; The final concentration of nicotinamide is 5-20 mM; The final concentration of A83-01 is 0.3-0.8 μM; The final concentration of Y-27632 is 5-20 μM.

5. The pancreatic cancer organoid culture medium according to claim 4, characterized in that The inhibitors also include gastrin; Preferably, in the pancreatic cancer organoid culture medium, the final concentration of gastrin is 5 to 20 nM.

6. The pancreatic cancer organoid culture medium according to claim 1, characterized in that The additives include one or more of 4-hydroxyethylpiperazineethanesulfonic acid, antibiotics and L-alanyl-L-glutamine; Preferably, in the pancreatic cancer organoid culture medium, the final concentration of the 4-hydroxyethylpiperazineethanesulfonic acid is 5 to 20 mM; The final concentration of the antibiotic is 1X; The final concentration of L-alanyl-L-glutamine was 2 mM.

7. The pancreatic cancer organoid culture medium according to claim 1, characterized in that The basic culture medium is Advanced DMEM / F12.

8. A method for culturing pancreatic cancer organoids, characterized in that: The following steps are involved: The pancreatic cancer tissue is mixed with a digestive fluid and subjected to a digestion process to obtain a digested tissue; The digested tissue was filtered through a cell strainer to obtain a precipitate; The precipitate is purified to obtain cells; The cells are inoculated into the pancreatic cancer organoid culture medium according to any one of claims 1 to 7, and the organoids are grown and cultured to obtain pancreatic cancer organoids.

9. The pancreatic cancer organoid culture method according to claim 8, characterized in that: The digestive fluid contains type 2 collagenase and neutral protease.

10. The pancreatic cancer organoid culture method according to claim 9, characterized in that: The final concentration of type 2 collagenase in the digestion solution is 1 to 3 mg / ml; The final concentration of the neutral protease in the digestive solution is 0.01-0.03 U / ml; And / or, the digestion treatment time is 1 to 2 hours.

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