Novel extraction method of primary breast cancer tumor-associated fibroblasts

Abstract

The invention discloses an extraction method of primary breast cancer tumor related fibroblasts, and belongs to the technical field of cell biology. The method provided by the invention can effectively solve the problems of long extraction time, easy pollution, small number of obtained cells and the like of the primary breast cancer tumor-related fibroblasts in the prior art, and can effectively improve the culture efficiency of the primary breast cancer tumor-related fibroblasts. In addition, by changing the formula of the digestive juice, the extraction time of primary cells is shortened, the pollution risk is reduced, meanwhile, the digestion efficiency is improved, the quantity of obtained cells is large, 0-generation cells can be placed in a T25 culture bottle, passage can be carried out in about 7 days, the culture period is shortened, and the method has important value for the economical efficiency of experimental research.

Description

Technical Field

[0001] The invention belongs to the technical field of cell biology, and particularly relates to a method for extracting primary breast cancer tumor-associated fibroblasts. Background Art

[0002] Breast cancer is the most common malignant tumor in Chinese women and the main cause of cancer-related death. Tumor-associated fibroblasts are activated fibroblasts and are the main components of the tumor microenvironment. They play an important role in promoting tumor growth, metastasis, drug resistance, immune barrier, etc. Therefore, the study of tumor-associated fibroblasts has become a hot spot in breast cancer research. However, the primary extraction of tumor-associated fibroblasts currently has the problems of long time consumption, easy pollution, and small number of cells obtained. The commonly used method is to digest the tissue with type I collagenase. The digestion process needs to be observed once every half an hour. The cells can be seen to swim out after about 2 hours of action. Generally, it is necessary to observe continuously for about 10 hours. The workload of the experimenter is large, and it is repeatedly taken and put in the incubator, which increases the risk of contamination. Therefore, this area is in urgent need of developing a new method for extracting primary breast cancer tumor-associated fibroblasts, so as to quickly extract and culture breast cancer tumor-associated fibroblasts. Summary of the invention

[0003] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for extracting primary breast cancer tumor-associated fibroblasts. The present invention shortens the primary cell extraction time and reduces the risk of contamination by changing the digestion solution formula, while improving the digestion efficiency, obtaining a large amount of cells, and the 0th generation cells can be placed in a T25 culture bottle and can be passaged in about 7 days, shortening the culture cycle.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0005] The present invention discloses a method for extracting primary breast cancer tumor-associated fibroblasts, characterized in that the method comprises: Step 1: Place the breast cancer tissue sample in PBS, trim excess fat and blood vessels, and wash repeatedly until there is no serum and fat; Step 2: Cut the tissue obtained in step 1 into minced meat; Step 3: Transfer the tissue obtained in step 2 to a centrifuge tube, add digestion solution 1 for digestion, filter through a filter, discard the filtrate, and retain the undigested tissue in the filter; Step 4: The undigested tissue obtained after filtration in step 3 is placed in a centrifuge tube, resuspended in digestion solution 2, and digested overnight; Step 5: After overnight culture in the culture flask obtained in step 4, the tissue is blown until the tissue disappears, centrifuged, the supernatant is discarded, and the tissue is resuspended in complete culture medium and then cultured; Step 6: Change the complete culture medium every 3 days, and on the 7th day, obtain primary breast cancer tumor-associated fibroblasts for subsequent passaging or purification experiments.

[0006] Furthermore, in step 3, the digestion solution 1 is composed of type II collagenase, hyaluronidase and DMEM / F12 culture medium.

[0007] Furthermore, in step 4, the digestion solution 2 is composed of type II collagenase, hyaluronidase, DNase, trypsin and DMEM / F12 culture medium.

[0008] Furthermore, in step 3, the digestion time of the digestion solution 1 is 60 minutes, and the digestion condition is a constant temperature shaker at 37°C.

[0009] Furthermore, in step 4, the digestion time of digestion solution 2 is overnight digestion, and the digestion conditions are 37°C, 5% CO 2 incubator.

[0010] Furthermore, the cell culture conditions in the method are 37°C and 5% CO 2 incubator.

[0011] The present invention also provides a primary breast cancer tumor-associated fibroblast extraction product, characterized in that the product comprises: digestive fluid 1, digestive fluid 2; The digestion solution 1 is composed of: type II collagenase, hyaluronidase and DMEM / F12 culture medium; The digestion solution 2 is composed of type II collagenase, hyaluronidase, DNase, trypsin and DMEM / F12 culture medium.

[0012] Furthermore, the above-mentioned product is used to extract and culture primary breast cancer tumor-associated fibroblasts in vitro.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0014] The method provided by the present invention is simple to operate and can effectively improve the culture efficiency of primary breast cancer tumor-associated fibroblasts. The method of the present invention can achieve excellent results exceeding traditional methods by using only easily available conventional reagents, and can achieve P0 cell culture in a relatively short time, which is worthy of promotion.

[0015] The present invention discloses a method for extracting primary breast cancer tumor-associated fibroblasts. By changing the formula of the digestion solution, the primary cell extraction time is shortened, the risk of contamination is reduced, and the digestion efficiency is improved. A large amount of cells are obtained. The 0th generation cells can be placed in a T25 culture flask and can be passaged in about 7 days, thereby shortening the culture cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The tissue was cut into minced meat and placed in a 50 mL centrifuge tube.

[0017] Figure 2 This is the precipitate obtained after the first centrifugation.

[0018] Figure 3 This is the microscopic finding after the first digestion using this method.

[0019] Figure 4 The remaining tissue after the first filtration was resuspended in digestion solution 2 and placed in a T25 culture flask for overnight digestion.

[0020] Figure 5 This is the precipitate obtained after the second centrifugation.

[0021] Figure 6 This is the microscopic finding after the second digestion using this method.

[0022] Figure 7 This is what we see under the microscope after one day of culture using this method.

[0023] Figure 8 This is what we saw under the microscope when changing the medium after 3 days of culture using this method.

[0024] Fig. 9 This is what we saw under the microscope when changing the medium after 6 days of culture using this method.

[0025] Fig.10 This method extracts the results of cell immunohistochemistry to identify α-SMA.

[0026] Fig.11 This method extracts the results of cell immunohistochemistry to identify Vimentin.

[0027] Fig.12 What is seen under the microscope on the first day of traditional culture.

[0028] Fig.13 Microscopic findings on the first day of culture using the improved method.

[0029] Fig.14 This method shows what was observed under the microscope on the first day of culture in the first bottle.

[0030] Fig.15 This method shows what was observed under the microscope on the first day of culture in the second bottle.

[0031] Fig.16 The findings under the microscope on the third day of traditional culture.

[0032] Fig.17 Microscopic findings on the third day of culture using the improved method.

[0033] Fig.18 This method shows what was observed under the microscope on the third day of culture in the first bottle.

[0034] Fig.19 This method shows what was observed under the microscope on the third day of culture in the second bottle.

[0035] Fig. 20 What we see under the microscope on the 6th day of culture using the traditional method.

[0036] Fig.21 Microscopic findings on the 6th day of culture using the improved method.

[0037] Fig. 22 This is what we saw under the microscope on the 6th day of culture in the first bottle of this method.

[0038] Fig.23 This is what we saw under the microscope on the 6th day of culture in the second bottle of this method.

[0039] Fig.24 This is what the second bottle of this method looked like on the 7th day of culture, ready for purification and passaging. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0041] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0042] Example 1: A method for extracting primary breast cancer tumor-associated fibroblasts.

[0043] The specific steps include: 1. One day before the experiment, coat the T25 culture flask with poly-lysine; 2. Fresh breast cancer tissue was removed during surgery and placed in PBS. After being transported to the laboratory on ice, excess fat and blood vessels were trimmed and washed repeatedly until there was no serum and fat. 3. Replace the instrument and cut the tissue obtained in step 2 into minced meat; 4. Transfer the tissue obtained in step 3 to a 50 mL centrifuge tube and add 5 mL of digestion solution 1 ( Figure 1 ) Digest in a 37°C constant temperature shaker for 60 min, use a Pasteur pipette to blow the tissue and then filter it through a 70 μm filter, discard the filtrate, and keep the undigested tissue in the filter; 5. Place the undigested tissue obtained in step 4 in a centrifuge tube, resuspend it in digestion solution 2, and place it in a T25 culture flask at 37°C and 5% CO. 2 Digest overnight in an incubator ( Figure 4 ); 6. Use a Pasteur pipette to blow the tissue digested overnight in the culture flask obtained in step 5 until the tissue disappears or almost disappears, and centrifuge at 252g for 10 minutes ( Figure 5), discard the supernatant, resuspend the precipitate with complete medium, place in a culture flask, and incubate at 37°C and 5% CO 2 Cultured in an incubator ( Figure 6 ); 7. After 1 day of culture, spindle cells can be seen ( Figure 7 ), the medium was changed every 3 days, and the number of spindle cells increased ( Figure 8 ), the cell mass was larger at 6 days ( Fig. 9 ).

[0044] The reagents in the above steps include.

[0045] 1. Digestion solution 1 (10 mL): 2.5 mL of type II collagenase (10 mg / mL) + 1 mL of hyaluronidase (10 mg / mL) + 6.5 mL of DMEM / F12 medium.

[0046] 2. Digestion solution 2 (10 mL): 2.5 mL of type II collagenase (10 mg / mL) + 1 mL of hyaluronidase (10 mg / mL) + 1 mL of DNase (10 mg / mL) + 5 mL of 0.25% trypsin + 0.5 mL of DMEM / F12 medium.

[0047] 3. Complete culture medium: DMEM / F12 medium, 10% fetal bovine serum, 5ng / mL fibroblast growth factor, 1% penicillin-streptomycin.

[0048] 4. PBS: The concentration purchased from the market is 1×PBS (pH 7.4).

[0049] Example 2: Comparative experiment on the effects of the extraction and cultivation method disclosed in the present invention.

[0050] 1. Reagents.

[0051] 1. Traditional method: Digest with 0.1 mg / mL type I collagenase overnight.

[0052] 2. Improved method: 0.01 mg / mL collagenase type II + 0.01 mg / mL hyaluronidase + 5% FBS digestion for 10 min.

[0053] 3. The method of the present invention: Digestive solution 1 (10 mL): type II collagenase (10 mg / mL) 2.5 mL + hyaluronidase (10 mg / mL) 1 mL + DMEM / F12 6.5 mL.

[0054] Digestion solution 2 (10 mL): 2.5 mL of type II collagenase (10 mg / mL) + 1 mL of hyaluronidase (10 mg / mL) + 1 mL of DNase (10 mg / mL) + 5 mL of 0.25% trypsin + 0.5 mL of DMEM / F12 medium.

[0055] 2. Operation method:

[0056] 1. Traditional method: Digestive fluid components: 0.1 mg / mL type I collagenase; Specific operation process: The tissue processing method is the same as the method of the present invention. The digestion solution is digested at 37°C for 10 hours, filtered, centrifuged, and the supernatant is discarded. The precipitate is resuspended with complete culture medium and placed in a culture bottle at 37°C and 5% CO 2 Culture in an incubator.

[0057] 2. Improvement method: Digestion fluid composition: 0.01 mg / mL type II collagenase + 0.01 mg / mL hyaluronidase + 5% FBS; Specific operation process: The tissue processing method is the same as the method of the present invention. The digestion solution is digested at 37°C for 10 minutes, filtered, centrifuged, and the supernatant is discarded. The precipitate is resuspended in complete culture medium and placed in a culture bottle at 37°C and 5% CO 2 Culture in an incubator.

[0058] 3. Comparative Example of Digestive Solution 1 of the Invention Method: Digestive fluid components: same as in Example 1; Specific operation process: The other processes are the same as those described in Example 1, except that: in step 3, after digestion and filtration of the digestion solution 1, centrifuge (252g) for 10 minutes to collect the filtrate ( Figure 2 ), discard the supernatant, resuspend the precipitate in complete culture medium, and place it in a culture flask at 37°C and 5% CO 2 Cultured in an incubator ( Figure 3 ), and obtained cells cultured only with digestion solution 1 (hereinafter referred to as “the first bottle of this method”).

[0059] 4. The method of the present invention: the process and reagents are the same as those described in Example 1 (hereinafter referred to as "the second bottle of the method").

[0060] 3. Experimental results.

[0061] 1. Number of digested cells: (Count cells according to existing technology).

[0062] Traditional method: 3×10 cells obtained 6 / mL.

[0063] Improved method: 10 cells were obtained 6 / mL.

[0064] Comparative Example of Digestion Solution 1 of the Present Invention (the First Bottle of the Present Method): 1.5×10 6 / mL.

[0065] The method of the present invention (the second bottle of the method): 4×10 cells were obtained 6 / mL.

[0066] It can be seen that the number of cells obtained by the method disclosed in the present invention is far superior to that obtained by other methods.

[0067] 2. Adhesion of tumor-associated fibroblasts.

[0068] a. First day (traditional method) Fig.12 , Improved method Fig.13 , the first bottle of this method Fig.14 , the second bottle of this method Fig.15 ): The traditional method and the improved method can hardly see the spindle cells. In this method, the first bottle needs to be carefully searched under the low-power field of view, and a small number of spindle cells can be seen. In this method, the spindle cells can be easily seen under the low-power field of view in the second bottle.

[0069] b. Day 3 (Traditional Method Fig.16 , Improved method Fig.17 , the first bottle of this method Fig.18 , the second bottle of this method Fig.19 ): The traditional method and the improved method seemed to show spindle cells, but they were not clear. In this method, the number of spindle cells in the first bottle increased slightly, and in the second bottle, the number of spindle cells increased significantly.

[0070] c. Day 6 (Traditional Method Fig. 20 , Improved method Fig.21 , the first bottle of this method Fig. 22 , the second bottle of this method Fig.23 ): A small amount of spindle cells can be seen under low-power microscope in both traditional and improved methods, with slightly more in the improved method than in the traditional method. In this method, the number of spindle cells in the first bottle increased, and in this method, the number of cells in the second bottle increased significantly, and the cells were relatively pure.

[0071] d. Day 7 (Second bottle of this method Fig.24 ): In this method, the amount of cells in the second bottle continued to increase, and some cells were observed to be arranged in a vortex shape, and then passage and purification were performed.

[0072] By comparison, it can be found that the second bottle of this method can quickly obtain a large number of cells for further passage, purification and subsequent experiments, and there is no bacterial contamination in the whole process.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting primary breast cancer tumor-associated fibroblasts, characterized in that: The method comprises: Step 1: Place the breast cancer tissue sample in PBS, trim excess fat and blood vessels, and wash repeatedly until there is no serum and fat; Step 2: Cut the tissue obtained in step 1 into minced meat; Step 3: Transfer the tissue obtained in step 2 to a centrifuge tube, add digestion solution 1 for digestion, filter through a filter, discard the filtrate, and retain the undigested tissue in the filter; Step 4: The undigested tissue obtained after filtration in step 3 is placed in a centrifuge tube, resuspended in digestion solution 2, and digested overnight; Step 5: After overnight culture in the culture flask obtained in step 4, the tissue is blown until the tissue disappears, centrifuged, the supernatant is discarded, and the tissue is resuspended in complete culture medium and then cultured; Step 6: Change the complete culture medium every 3 days, and on the 7th day, obtain primary breast cancer tumor-associated fibroblasts for subsequent passaging or purification experiments.

2. The extraction method according to claim 1, characterized in that The digestion solution 1 in step 3 is composed of type II collagenase, hyaluronidase and DMEM / F12 culture medium.

3. The extraction method according to claim 1, characterized in that The digestion solution 2 in step 4 is composed of type II collagenase, hyaluronidase, DNase, trypsin and DMEM / F12 culture medium.

4. The extraction method according to claim 1, characterized in that In step 3, the digestion time of digestion solution 1 is 60 minutes, and the digestion condition is a constant temperature shaker at 37°C.

5. The extraction method according to claim 1, characterized in that In step 4, the digestion time of digestion solution 2 is overnight digestion, and the digestion conditions are 37° C. and 5% CO 2 in an incubator.

6. The extraction method according to claim 1, characterized in that The cell culture conditions in the method are culturing in a 37° C., 5% CO 2 incubator.

7. A primary breast cancer tumor-associated fibroblast extraction product, characterized in that: The products include: digestive juice 1, digestive juice 2; The digestion solution 1 is composed of: type II collagenase, hyaluronidase and DMEM / F12 culture medium; The digestion solution 2 is composed of type II collagenase, hyaluronidase, DNase, trypsin and DMEM / F12 culture medium.

8. The product according to claim 7 is used for extracting and culturing primary breast cancer tumor-associated fibroblasts in vitro.

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