CN104312976A - Separating method of tumor stem cells - Google Patents

Abstract

The invention discloses a method for isolating tumor stem cells. The method comprises: digesting tumor cells into individual cells, adding digestive enzyme solution and incubating for several hours; adding the incubated cells into cell culture solution containing serum for adherent growth; After the cells adhered to the wall, the tumor stem cell TSC medium was replaced for culture; when the cells were full, the above operations were repeated several times, and the obtained cells were cultured in microspheres, and the finally obtained microspheres were tumor stem cells. For the first time, the present invention uses a variety of digestive enzymes or combined with other physical stress conditions to stimulate a variety of tumor cells, and can stably obtain different types of TSCs; the isolated MuseTSCs of the present invention have strong tumor stemness, good homogeneity, and stability. Therefore, the establishment of this method can standardize the acquisition of different types of TSCs, and is more simple and economical. It is suitable for large-scale construction of TSC libraries to meet the needs of routine tumor biomedical basic research, clinical research, and drug development.

Description

A method for isolating tumor stem cells

technical field

The invention belongs to the technical field of tumor stem cells, and relates to a method for separating tumor stem cells.

Background technique

In recent years, a large number of reports have found that there are a very small number of cells in the tumor, which are extremely malignant. When the patient undergoes radiotherapy and chemotherapy, the large tumor mass disappears immediately. However, these very small number of cells can still survive, and PET-CT cannot detect them. Existence, and it is these malignant cells that become more ferocious after relapse, a large number of metastases devour life, these cells are tumor stem cells (tumor stem cells, tumor stem cells, TSC; or cancer stem cells, cancer stem cells, CSC; tumor initiating cells , tumor initiating cell, TIC; cancer initiating cell, cancer initiating cell, CIC), many mainstream views today believe that tumor stem cells enriched by radiotherapy and chemotherapy are the main reason for the failure of traditional treatment methods. In 2006, the American Association for Cancer Research defined TSC as: cells in tumors that have self-renewal ability and can differentiate to produce heterogeneous tumor cells. Its self-renewal ability and differentiation ability fully meet the two main characteristics of stem cells, while These two characteristics are the necessary reasons for tumor recurrence and tumor metastasis. Ordinary tumor cells need hundreds of thousands to millions of cells to form tumors in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, while TSC only needs tens to hundreds of cells to form tumors. Even 1-10 TSCs can form tumors, which shows their extremely high tumorigenicity and malignancy. Therefore, solving the problem of TSCs is one of the most important directions for future cancer research and treatment.

In 1997, through the study of acute myeloid leukemia stem cells, Bonnet et al. proved that most leukemia cells could not continue to proliferate, and only a few cells with a phenotype of CD34 ⁺ CD38 ^- could form tumors in NOD/SCID mice, which was confirmed for the first time the existence of TSC. In 2003, AlHajj et al. isolated breast cancer cells with a phenotype of CD44 ⁺ CD24 ^-/low Lineage from breast cancer tissue, which was the first time that solid tumor stem cells were isolated. So far, TSCs have been isolated from various tumors such as leukemia, brain tumor, lung cancer, prostate cancer, colon cancer, liver cancer, nasopharyngeal cancer, pancreatic cancer, melanoma, ovarian cancer, and prostate cancer.

Although more and more evidence supports the TSC theory, there are still many problems in the research of TSC: first, not all tumors have isolated TSC; second, the technology of TSC isolation and identification is still not perfect; third, there are still many problems. There is no definite TSC-specific surface marker; Fourth, the origin, occurrence, and molecular and cellular mechanisms of TSC have not been deeply studied, and there are still many major issues to be resolved. To solve these problems, a large number of TSCs are needed as the basis, and there is no institution specialized in providing various TSCs in the world. It can be seen that the source of TSCs is very scarce. Usually, most of the TSCs needed for basic research or clinical research are self-made. It takes a long time to construct or contact the author of the report literature to obtain it, the cost is high, and the variety is not complete, so there are many restrictions. Due to the imperfect and difficult technology of TSC separation and identification, the pace of TSC-related research has been further hindered. It is imminent to establish a TSC preparation method with high versatility, easy operation and suitable for large-scale library construction.

The primary problem in constructing a TSC cell bank is to isolate TSCs from tumor cells or tumor tissues. Since the proportion of TSCs in tumor tissues is very small, there are still many types of tumor TSCs that have not been isolated so far. At present, there are three main separation methods of TSC:

1. TSC-specific surface marker method : According to the difference between the surface markers of TSC and ordinary tumor cells, by flow cytometry (fluorescence activated cell sorting, FACS) or immunomagnetic bead sorting (magnetic activated cell sorting, MACS) to obtain TSC. Dalerba et al. used CD44 and epithelial cell adhesion molecule EpCAM as markers, and successfully sorted colon cancer stem cells with a phenotype of EpCAM ^high CD44 ⁺ by FACS. Yuan et al. successfully isolated CD133 ⁺ glioma stem cells from human glioma using MACS. However, these two separation techniques both rely on specific markers on the surface of TSCs, and there is still a lack of effective TSC surface markers, resulting in many types of tumors without reliable surface markers that cannot be sorted into TSCs by this method. In addition, this method The instruments and reagents used are extremely expensive, and the requirements for experimenters are quite high, which is not suitable for large-scale sorting of TSCs of various types of tumors.

2. SP cell separation method : SP cells are a group of stem cells that cannot be stained by the DNA dye Hoechst33342. These cells highly express cell surface transport proteins such as ABC transporters, and can discharge Hoechst 33342 out of the cells without staining. These cells can excrete chemotherapy drugs out of the cells, have drug resistance and TSC biological characteristics, and have stronger invasiveness and tumorigenic ability than non-SP cells. SP-TSC can also be isolated by flow cytometry. At present, SP-TSCs have been isolated from various tumor tissues such as glioma, lung cancer, and breast cancer. However, SP-TSC is not a group of homogeneous cells, and the heterogeneity is still very strong. Some SP cells do not have complete stem cell characteristics, and not all types of tumor tissue cells can isolate SP-TSC with stemness. , and this method still requires flow sorting operations, and also requires expensive sorting-level flow cytometers, and is also not suitable for large-scale sorting of TSCs.

3. Spheroids suspension culture method : This method is a method for isolating and obtaining TSC by using the characteristic that TSC can grow in suspension in the form of single cells to form microspheres (Spheroids) in serum-free medium. Singh et al. used this method to obtain CD133+ glioma stem cells, and Ponti et al. also obtained CD44+CD24-Cx43- breast cancer stem cell spheres. However, not all types of tumor cells can form Spheroids in suspension in a single-cell state, only stem cells with self-renewal ability can form Spheroids, and the tumorigenicity of Spheroids obtained without other methods is not always However, the formation of Spheroids can intuitively reflect the self-renewal ability of cells, and is also widely used in the identification, enrichment and purification of tumor stem cells.

The existence of TSC is one of the most fundamental reasons for the recurrence of malignant tumors. At present, there is no effective drug targeting TSC. Therefore, in-depth research on TSC will help us master new technologies and find more effective means of tumor diagnosis and treatment. The research on TSC must have the means to obtain TSC, and the current methods of obtaining TSC are uneven, so it is of great significance to establish a new simple and high-quality TSC acquisition method.

In 2010, Japanese scientist Mari Dezawa found that there is a subset of cells with the ability to resist exogenous stress in the cell population of mesenchymal origin (such as mesenchymal stem cells and fibroblasts), and these cells can tolerate digestive enzymes (reported 0.25% trypsin), low oxygen, no serum, no nutrients and other stress stress stimulation without death, and these cells have a stronger differentiation ability than ordinary mesenchymal cells, and can differentiate into three germ layers Differentiation, mesenchymal cells other than this subset are unable to differentiate accordingly and do not form teratomas like other pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells, which is not the case with other discovered adult stem cells These cells are called multilineage differentiating stress enduring cells (Muse Cells) (Proc Natl Acad Sci U S A. 2010 May 11;107(19):8639-43. ). It was later found that the double-positive cells of SSEA3 (surface marker of pluripotent stem cells) and CD105 (surface marker of mesenchymal cells) in mesenchymal cells had typical characteristics of Muse cells, and could differentiate into three germ layers without forming teratomas. The selected method can better obtain Muse cells with great regenerative medicine application value and strong differentiation ability (Nat Protoc. 2013;8(7):1391-415.), these cells are also a good source of induced pluripotent stem cells (Proc Natl Acad Sci U S A. 2011 Jun 14;108(24):9875-80.). It seems that there is a group of cells in the mesenchymal population that can withstand external physical pressure and have stronger self-renewal and differentiation capabilities, and TSC is a type of tumor cells with stronger self-renewal and differentiation capabilities, but each There is no uniform screening surface marker for different types of TSC, and its source is far more than mesenchymal type cells, including a large number of epithelial, neural, lymphoid, blood and other types of tumors, and it is difficult to effectively separate them by using a single unified molecular marker for sorting Various types of TSCs.

Contents of the invention

In order to solve the above-mentioned problems, the present invention establishes a novel, reliable and convenient separation method for tumor stem cells (TSCs), which can obtain TSCs from various types of tumors, to create a public TSC cell bank platform, and to fill TSC research resources The lack of technology provides a solid technical foundation.

The purpose of the present invention is to provide a method for isolating tumor stem cells.

The technical scheme that the present invention takes is:

A method for isolating tumor stem cells, comprising the steps of:

1) After digesting the adherent tumor cells to a single cell with digestive enzyme solution 1, stop the digestion, centrifuge, and collect the precipitated cells;

2) Add digestive enzyme solution 2 to the collected cells and incubate for 0.5-10 hours;

3) Wash the incubated cells, centrifuge, collect the precipitated cells, add serum-containing cell culture medium, mix well, inoculate the cells into cell culture dishes for adherent growth;

4) After the above cells adhered to the wall, the tumor stem cell TSC medium was replaced for culture;

5) When the cells are full, repeat steps 1) to 4) 2 to 6 times, and the obtained cells are called Muse-tumor cells;

6) Spheroids culture: Inoculate the Muse-tumor cells obtained above into a cell culture dish, and use TSC medium to make the Muse-tumor cells grow single-cell and low-density adherent. When most cells form uniform and dense clones, the nucleus and cytoplasm When the ratio increases and has typical stem cell characteristics, remove a small number of heterogeneous non-tumor stem cells, digest the overgrown Muse-tumor cells into single cells, and use TSC medium to suspend to agarose-matched or low-adhesion cells Suspension culture in a petri dish, the obtained microsphere Spheroids are tumor stem cells, called Muse TSC.

Further, a method for isolating tumor stem cells, comprising the following steps:

1) After digesting the adherent tumor cells to a single cell with digestive enzyme solution 1, stop the digestion, centrifuge, and collect the precipitated cells;

2) Add digestive enzyme solution 2 to the collected cells and incubate at 1-37°C for 0.5-10 hours to simulate the stress state of digestive enzymes;

3) Move the above-incubated cells to 1-18°C and continue to incubate for 10-72 hours to simulate the low temperature and digestive enzyme stress state;

4) Wash the incubated cells, centrifuge, collect the precipitated cells, add serum-free or serum-less than 5% (v/v) cell culture medium, mix well, and store at 1-37°C under airtight conditions Incubate for 20-70 hours under low temperature to simulate the state of serum-free and hypoxic stress;

5) Centrifuge the cells incubated in the previous step, collect the precipitated cells, add cell culture medium containing 1-20% (v/v) FBS, mix well, inoculate cells into cell culture dishes for adherent growth;

6) After the above cells adhered to the wall, the tumor stem cell TSC medium was replaced for culture;

7) When the cells are full, repeat steps 1) to 6) 2 to 6 times, and the obtained cells are called Muse-tumor cells;

8) Spheroids culture: Inoculate the Muse-tumor cells obtained above into cell culture dishes, and use TSC medium to make the Muse-tumor cells grow single-cell and low-density adherent. When most cells form uniform and dense clones, the nucleus and cytoplasm When the ratio increases and has typical stem cell characteristics, remove a small number of heterogeneous non-tumor stem cells, digest the overgrown Muse-tumor cells into single cells, and use TSC medium to suspend them in agarose-matched cell culture dishes. Suspension culture, the obtained microsphere Spheroids are tumor stem cells, called Muse TSC.

Further, the digestive enzyme in the above-mentioned digestive enzyme solution 1 is selected from one of trypsin, TrypLE, and Accutase.

Further, 0.2-15 mL of digestive enzyme solution 2 is added to every 1×10 ⁶ to 9×10 ⁶ cells in the above step 2).

Further, the above digestive enzyme solution 2 is selected from 0.02-0.3% (w/v) type I collagenase solution, 0.02-0.3% (w/v) type IV collagenase solution, 0.1-3U/ml Dispase dispersion Enzyme solution, 0.01-0.3% (w/v) trypsin solution, 0.01-1% (w/v) TrypLE enzyme solution, 0.01-1% (w/v) Accutase enzyme solution.

Further, for all the cell culture solutions mentioned above, different cell basal media are selected according to different types of tumor cells.

Further, in the above Spheroids culture, the number of Muse-tumor cells seeded into the cell culture dish is 100-2000 cells per well of the six-well plate.

Further, the Muse TSC obtained above was digested into single cells and cultured in suspension again no less than once to obtain higher purity, smoother and more stable microsphere Spheroids, that is, Muse TSC.

The beneficial effects of the present invention are:

1) For the first time, the present invention uses a variety of digestive enzymes (including type I collagenase, type IV collagenase, dispase, trypsin, TrypLE, Accutase), or combined with other physical stress conditions to stimulate a variety of tumor cells, and can stably obtain different Type of TSC, called Muse TSC, through identification, the Muse TSC separated by the present invention has extremely strong tumor stemness, good homogeneity, relatively stable, and can repeatedly obtain suspension culture microspheres (Muse cells) derived from single cells. -Spheroids), so the establishment of this method can standardize the acquisition of different types of TSCs, and is more simple and economical, suitable for large-scale construction of TSC libraries to meet the needs of routine tumor biomedical basic research, clinical research, and drug development.

2) The present invention is original. The present invention improves the isolation method of multi-lineage differentiated cells under continuous stress, and applies it to the isolation of tumor stem cells in combination with the traditional Spheroids culture method; this invention uses a new and unique culture method for the first time Various types of tumor stem cells have been isolated, and these tumor stem cells are stronger than those obtained by traditional separation and sorting methods in terms of quality, purity and stemness. The method is simple and economical, does not require harsh experimental conditions, and does not Expensive experimental equipment is required, and it is suitable for the establishment of a large number of types of tumor stem cell banks.

3) The present invention establishes a low-cost, fast, and convenient method for obtaining various types of tumor stem cells. The obtained tumor stem cells are called Muse TSCs. After strict identification, their tumor stemness and malignancy are much higher than those obtained by traditional methods. The method of the invention has the ability to construct multiple types of tumor stem cells in large quantities at the same time.

4) The present invention provides a solid technical basis for establishing standardized, compatible, universal, high-quality, and open tumor stem cell banks derived from various types of tumors and filling the lack of high-end tumor biology resource banks; the tumor stem cells prepared by the method of the present invention It will cover all levels of research in tumor biomedicine, and provide reliable technical resources for clinical diagnosis and treatment of tumors, basic research, and new drug development in my country.

5) The present invention will provide high-quality tumor stem cells for scientific research and clinical workers. These abundant and diverse tumor stem cells will be used for in-depth exploration of basic scientific research, research and development and breakthrough of early malignant tumor diagnosis, and specific tumor stem cell targeting. Screening of small molecule compounds, research and development of gene therapy drugs targeting tumor stem cells, research and development of bio-missiles targeting tumor stem cell humanized antibodies, development of specific immune cell therapy technology targeting tumor stem cells, research and development of a new generation of radiotherapy and chemotherapy technologies for preventing tumor recurrence etc. Therefore, the method of the present invention will benefit the clinical and basic cancer research in the region and even the whole country, and advance the prevention and treatment of cancer to a new stage.

Description of drawings

Figure 1 is a low-density single-cell adherent culture image of 3 ^rd -Muse-HepG2 under a 100-fold microscope; the double arrows point to the formation of most uniform and dense clones with an increased nuclear-to-cytoplasmic ratio, which is significantly different from the original HepG2 morphology. It has typical stem cell characteristics; and the single arrow points to a very small number of clones that maintain the original cell shape of HepG2;

Figure 2 is a picture of HepG2-Muse TSC microspheres under a 40x microscope. The 3 ^rd -Muse-HepG2 was digested into single cells, suspended and seeded in an agarose-lined well plate for culture, and then purified three times. Spheroids are HepG2-Muse TSC;

Figure 3 is the results of FACS detection of TSC surface marker CD133 in ordinary HepG2 (A) and HepG2-Muse TSC (B) cells (green is the peak map of CD133 antibody, red is the peak map of the corresponding isotype control (Isotype Control) antibody);

Fig. 4 is the formation situation of common HepG2 cell and HepG2-Muse TSC cell prepared in embodiment 1 in the low concentration serum condition monoclonal;

Figure 5. The left picture shows that 10,000 HepG2-Muse TSC cells formed obvious Spheroids on the fifth day in low-concentration serum medium (2% FBS), and the right picture shows that HepG2 under the same conditions did not form obvious Spheroids on the tenth day Spheroids (observed with a 40x lens);

Fig. 6 is a tumor figure taken out by dissecting each group of mice in Table 1;

Figure 7 is the Hep3B-Muse TSC (liver cancer stem cell) prepared in Example 2;

Figure 8 is the results of FACS detection of TSC surface marker CD133 in ordinary Hep3B (A) and Hep3B-Muse TSC (B) cells (green is the peak map of CD133 antibody, red is the peak map of the corresponding isotype control (Isotype Control) antibody);

Figure 9 is the U251-Muse TSC (glial tumor stem cell) prepared in Example 3;

Figure 10 is the detection results of the tumor stemness marker CD133 of common U251 (A) and U251-Muse TSC (B) prepared in Example 3 (green is the CD133 antibody peak map, red is the corresponding isotype control (Isotype Control) antibody peak picture);

Figure 11 shows that 1000 U251, U251 spheroids-TSC and U251-Muse TSC cells were planted in the medium containing 10% FBS, 2% FBS and 0.5% FBS respectively, and the crystal violet stained cell clones were cultured for 9 days after single-cell adhesion ;

Figure 12 is the MCF7-Muse TSC (breast tumor stem cell) prepared in Example 4;

Figure 13 is the detection results of the tumor stemness marker CD133 of MCF7 (A) and MCF7-Muse TSC (B) prepared in Example 4 (green is the CD133 antibody peak map, red is the corresponding isotype control (Isotype Control) antibody peak map ).

Detailed ways

A method for isolating tumor stem cells:

1) After digesting the adherent tumor cells to a single cell with digestive enzyme solution 1, stop the digestion, centrifuge, and collect the precipitated cells;

2) Add digestive enzyme solution 2 to the collected cells and incubate for 0.5-10 hours;

3) Wash the incubated cells, centrifuge, collect the precipitated cells, add serum-containing cell culture medium, mix well, inoculate the cells into cell culture dishes for adherent growth;

4) After the above cells adhered to the wall, the tumor stem cell TSC medium was replaced for culture;

5) When the cells are full, repeat steps 1) to 4) 2 to 6 times, and the obtained cells are called Muse-tumor cells;

6) Spheroids culture: Inoculate the Muse-tumor cells obtained above into a cell culture dish, and use TSC medium to make the Muse-tumor cells grow single-cell and low-density adherent. When most cells form uniform and dense clones, the nucleus and cytoplasm When the ratio increases and has typical stem cell characteristics, remove a small number of heterogeneous non-tumor stem cells, digest the overgrown Muse-tumor cells into single cells, and use TSC medium to suspend to agarose-matched or low-adhesion cells Suspension culture in a petri dish, the obtained microsphere Spheroids are tumor stem cells, called Muse TSC.

Preferably, the above-mentioned method for isolating tumor stem cells comprises the following steps:

1) After digesting the adherent tumor cells to a single cell with digestive enzyme solution 1, stop the digestion, centrifuge, and collect the precipitated cells;

2) Add digestive enzyme solution 2 to the collected cells and incubate for 0.5-10 hours. The preferred incubation temperature is 1-37°C, most preferably 37°C, to simulate the stress state of digestive enzymes;

3) Move the above-incubated cells to 1-18°C and continue to incubate for 10-72 hours to simulate the low temperature and digestive enzyme stress state; the temperature is preferably 2-6°C, most preferably 4°C, and the incubation time is preferably 12～16h.

4) Wash and centrifuge the incubated cells, collect the precipitated cells, add serum-free or serum less than 5% (v/v), mix well, and incubate for 20-70 h under airtight conditions, preferably at 1 Incubate at ~37°C for 40-60h, the most preferred incubation temperature is 37°C, simulating the state of serum-free and hypoxic stress;

5) Centrifuge the cells incubated in the previous step, collect the precipitated cells, add the cell culture medium containing 1-20% (v/v) FBS, the FBS concentration is most preferably 2%, mix well, and inoculate the cells into the cell culture Adhesive growth in a dish;

6) After the above cells adhered to the wall, the tumor stem cell TSC medium was replaced for culture;

7) When the cells are full, repeat steps 1) to 6) 2 to 6 times (preferably 3 to 4 times), and the obtained cells are called 3 ^rd /4 ^th -Muse-tumor cells;

8) Spheroids culture: Inoculate the Muse-tumor cells obtained above into cell culture dishes, and use TSC medium to make the Muse-tumor cells grow single-cell and low-density adherent. When most cells form uniform and dense clones, the nucleus and cytoplasm When the ratio increases and has typical stem cell characteristics, remove a small number of heterogeneous non-tumor stem cells, digest the overgrown Muse-tumor cells into single cells, and use TSC medium to suspend to agarose-matched or low-adhesion cells Carry out suspension culture in a petri dish (preferably agarose-matched cell culture dish suspension culture), and the obtained microsphere Spheroids are tumor stem cells, called Muse TSC.

Preferably, the concentration of the digestive enzyme contained in the digestive enzyme liquid 1 is 0.25% (w/v), and the digestion time is 2-5 minutes, and the digestion time is related to different types of tumor cells.

Preferably, the digestive enzyme in the above-mentioned digestive enzyme solution 1 is selected from one of trypsin, TrypLE, and Accutase, most preferably trypsin.

Preferably, 0.2-15 mL of digestive enzyme solution 2 is added to every 1×10 ⁶ to 9×10 ⁶ cells in the above step 2), most preferably 1 mL.

Preferably, the time for incubating the cells with the digestive enzyme solution 2 in the above step 2) is 4-8 hours, most preferably 6 hours.

Preferably, the digestive enzyme solution 2 is selected from 0.02-0.3% (w/v) type I collagenase solution, 0.02-0.3% (w/v) type IV collagenase solution, and 0.1-3 U/ml Dispase dispersion Enzyme solution, 0.01-0.3% (w/v) trypsin solution, 0.01-1% (w/v) TrypLE enzyme solution, 0.01-1% (w/v) Accutase enzyme solution, most preferably 0.02-0.3% (w/v) type I collagenase solution.

Preferably, all the above-mentioned cell culture solutions select different cell basal media according to different types of tumor cells.

Preferably, all of the above TSC culture media are selected from different TSC culture media according to different types of tumor cells.

Preferably, in the above Spheroids culture, the number of Muse-tumor cells seeded into the cell culture dish is 100-2000 cells per well of the six-well plate.

Preferably, the Muse TSC obtained above is digested into single cells and cultured in suspension no less than once again to obtain higher purity, smoother and more stable microsphere Spheroids, that is, Muse TSC.

The present invention will be further described below in conjunction with specific examples, but is not limited thereto.

The cell lines of liver cancer, breast cancer, and glioma used in the following are all from the Cell Biology Experimental Bank of Guangzhou Boxer Cancer Institute.

Example 1 The separation method of HepG2 liver tumor stem cells

1) Digest HepG2 adherent hepatoma cells with 0.25% trypsin solution at 37°C for 2-5 minutes to single cells, terminate with medium containing serum, centrifuge at 200g/min for 5min, and collect the precipitated cells; (here Steps can use other digestive enzymes instead of trypsin solution, such as TrypLE, Accutase, etc., the digestion time, digestion temperature, and centrifugation time are related to different types of tumor cells);

2) Add 0.1% type I collagenase solution (diluted with basal medium DMEM/F12) to the collected cells above, add 1mL enzyme solution per 1× ¹⁰⁶ cells, and incubate at 37°C for 6h to simulate digestive enzymes Stress state; (This step can use other digestive enzymes to replace 0.1% type I collagenase solution, such as 0.02-0.3% (w/v) type IV collagenase solution, 0.1-3U/ml Dispase solution, 0.01-0.3% (w/v) trypsin solution, 0.01-1% (w/v) TrypLE enzyme solution, 0.01-1% (w/v) Accutase enzyme solution, etc. Other digestive enzymes are also used in this invention Within the protection range, the most preferred is 0.1% (w/v) type I collagenase solution; the incubation time can be 0.5-10h, preferably 4-8h, most preferably 6h;)

3) Move the above-incubated cells to 4°C and incubate for 12-16 hours to simulate low temperature and digestive enzyme stress state; (The temperature for simulating low temperature stress state can be 1-18°C, preferably 2-6°C, most preferably 4°C; the incubation time can be 10~72h, preferably 12~16h;)

4) Wash the incubated cells with PBS buffer, mix well, centrifuge at 200g/min for 5min, discard the supernatant, wash away the enzyme solution, add DMEM/F12 culture medium to the cells precipitated in the centrifuge tube until the centrifuge tube is full , cover the cap of the centrifuge tube, seal the bottle with a parafilm, mix it upside down, and incubate it in a 37°C incubator for 48 hours to simulate the state of serum-free and hypoxic stress; (steps 3) to 4) can be omitted, Directly perform the operation of step 5) on the cells incubated in step 2), but the optimal treatment method is to include steps 3) to 4);)

5) Centrifuge the cells incubated in the previous step at 200g/min for 5min, collect the precipitated cells, add DMEM/F12 culture medium containing 2% (v/v) FBS, mix well, and inoculate the cells into tissue culture-treated Adhesive growth in ordinary cell culture dishes;

6) After the above cells adhered to the wall, the liver cancer TSC medium was replaced for culture, and the liver cancer TSC medium was DMEM containing 20ng/ml EGF, 10 ng/ml bFGF, 2% B27 supplement without vitamin A (Invitrogen), and 1% N2 supplement /F12 medium;

7) When the cells are congested, repeat steps 1) to 6) three times, and the obtained cells are called 3 ^rd -Muse-HepG2;

8) Spheroids culture: Inoculate the 3 ^rd -Muse-HepG2 obtained above into a tissue culture-treated cell culture dish (6-well plate contains 100-2000 cells per well), and use liver cancer TSC medium at a low density of single cells Adhering to the wall, most of the cells formed uniform and dense clones after a few days, and the nuclear-to-cytoplasmic ratio increased, which was obviously different from the original HepG2, and had typical stem cell characteristics. Qualified non-TSCs were scraped off with a scraper needle (see Figure 1); the overgrown 3 ^rd -Muse-HepG2 was digested into single cells, and the liver cancer TSC medium was used to suspend them into agarose-lined cell culture dishes for suspension culture , the obtained Spheroids (microspheres) are liver tumor stem cells, called HepG2-Muse TSC, and then repeated 3 times for purification into single cells and suspension culture to obtain smoother and more stable HepG2-Muse TSC (see Figure 2 );

The undifferentiated HepG2-Muse TSC prepared in Example 1, which is capable of stably forming single-cell spheres for passage, is identified by surface markers (FACS identification), and repeated single-cell sphere-forming and tumor-forming experiments of a very small amount of TSC confirm that it has significant tumor stemness.

(1) Identification by flow cytometry (FACS)

Using FACS to identify the tumor stem surface marker CD133 in common HepG2 and HepG2-Muse TSC cells prepared in Example 1, the results showed that more than 96% of HepG2-Muse TSC had CD133 markers, while less than 0.1% of common HepG2 cells contained CD133 ( As shown in Figure 3).

(2) Low serum concentration colony formation assay

100, 1000, 3000 common HepG2 cells and HepG2-Muse TSC cells prepared in Example 1 were respectively planted in a six-well plate, and then adhered monoclonal culture was carried out using a low-concentration serum medium containing 2% FBS , and observed colony formation after one week by staining with crystal violet.

The results of clone formation experiments found that only 100 cells of HepG2-Muse TSC in a six-well plate can form obvious clones in medium with low concentration of serum (2% serum concentration), while ordinary HepG2 needs nearly 3000 cells to form relatively Obvious cloning (see Figure 4).

(3) Spheroids culture experiment

Take 10,000 ordinary HepG2 cells and HepG2-Muse TSC cells prepared in Example 1 to culture Spheroids in low-concentration serum medium containing 2% FBS, and observe the formation of Spheroids (microspheres).

The present invention finds that 10,000 HepG2-Muse TSC cells can form obvious Spheroids in the low-concentration serum medium on the fifth day through the Spheroids culture experiment, while 10,000 HepG2 cells have not formed obvious Spheroids until the tenth day (see figure 5).

(4) Tumorigenicity detection

Inoculate 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 5X10 ⁵ , 10 ⁶ , 2X10 ⁶ HepG2 cells and HepG2-Muse TSC cells prepared in Example 1 into NOD-SCID mice subcutaneously, one month later It was found that 2 of the 5 mice inoculated with 10 HepG2-Muse TSC cells formed tumors, and all the mice inoculated with 100 or more HepG2-Muse TSC cells formed tumors, while ordinary HepG2 cells were inoculated with 500,000 cells. Only 3 of the 5 mice with cells formed tumors, and the inoculated amount of less than 500,000 failed to form tumors ( see Figure 6 and Table 1 ). It can be seen that the HepG2-Muse TSC prepared by the present invention has extremely strong self-renewal ability and Tumorigenicity, with relatively complete TSC characteristics.

Table 1 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 5×10 ⁵ , 10 ⁶ , 2×10 ⁶ HepG2 and HepG2-Muse TSC cells were each inoculated subcutaneously in 5 NOD-SCID mice , Count the number of tumor mice in each group after one month.

Table 1 The results of tumor formation experiments in mice

 

Example 2 The separation method of Hep3B liver tumor stem cells

1) Use 0.25% trypsin solution to digest Hep3B adherent hepatoma cells at 37°C for 2-5 minutes to form a single cell, stop with medium containing serum, centrifuge at 200g/min for 5min, and collect the precipitated cells;

2) Add 0.1% type I collagenase solution (diluted with basal medium DMEM/F12) to the collected cells above, add 1mL enzyme solution per 1× ¹⁰⁶ cells, and incubate at 37°C for 6h to simulate digestive enzymes Stress state;

3) Move the above-incubated cells to 4°C and incubate for 12-16 hours to simulate low temperature and digestive enzyme stress state;

4) Wash the incubated cells with PBS buffer, mix well, centrifuge at 200g/min for 5min, discard the supernatant, wash away the enzyme solution, add DMEM/F12 culture medium to the cells precipitated in the centrifuge tube until the centrifuge tube is full , cover the cap of the centrifuge tube, seal the bottle mouth with a parafilm, mix it upside down, and incubate it in a 37°C incubator for 48 hours to simulate the state of serum-free and hypoxic stress;

5) Centrifuge the cells incubated in the previous step at 200g/min for 5min, collect the precipitated cells, add DMEM/F12 culture medium containing 2% (v/v) FBS, mix well, and inoculate the cells into tissue culture-treated Adhesive growth in ordinary cell culture dishes;

6) After the above cells adhered to the wall, the liver cancer TSC medium was replaced for culture, and the liver cancer TSC medium was DMEM containing 20ng/ml EGF, 10 ng/ml bFGF, 2% B27 supplement without vitamin A (Invitrogen), and 1% N2 supplement /F12 medium;

7) When the cells are full, repeat steps 1) to 6) 4 times, and the obtained cells are called 4 ^th -Muse-Hep3B;

8) Spheroids culture: Inoculate the 4 ^th -Muse-Hep3B obtained above into a tissue culture-treated cell culture dish (6-well plate contains 100-2000 cells per well), and use liver cancer TSC medium to grow at a low density of single cells Adhering to the wall, after a few days, most cells formed uniform and dense clones with increased nuclear-to-cytoplasmic ratios, which were significantly different from the original Hep3B in shape, and had typical stem cell characteristics, while a very small number of clones maintained the original cell shape of Hep3B, and the heterogeneous The qualitative non-TSC was scraped off with a scraper needle; the overgrown 4 ^th -Muse-Hep3B was digested into single cells, and the liver cancer TSC medium was used to suspend them in agarose-lined cell culture dishes for suspension culture, and the obtained Spheroids ( Microspheres) are hepatic tumor stem cells, called Hep3B-Muse TSC, and the purification process of digestion into single cells and suspension culture was repeated twice to obtain a smoother and more stable Hep3B-Muse TSC (see Figure 7);

The Hep3B-Muse TSC prepared in Example 2 is identified below.

Flow Cytometry (FACS) Identification

FACS was used to identify the tumor stem surface marker CD133 in ordinary Hep3B cells (Figure 8A) and liver cancer Hep3B-Muse TSCs prepared in Example 2 (Figure 8B). About 0.6% of Hep3B cells contain CD133 (as shown in Figure 8).

Example 3 Separation method of glial tumor stem cells

1) Digest adherent glioma cells U251 with 0.25% trypsin solution at 37°C for 4 minutes to single cells, terminate with medium containing serum, centrifuge at 200g/min for 5min, and collect the precipitated cells;

2) Add 0.1% type I collagenase solution (prepared from DMEM culture medium) to the collected cells above, add 1mL enzyme solution per 1× ¹⁰⁶ cells, incubate at 37°C for 6h to simulate digestive enzymes Stress state;

3) Move the above-incubated cells to 4°C and incubate for 12-16 hours to simulate low temperature and digestive enzyme stress state;

4) Wash the incubated cells with PBS buffer, mix well, centrifuge at 200g/min for 5min, discard the supernatant, wash off the enzyme solution, add DMEM culture medium to the precipitated cells in the centrifuge tube until the centrifuge tube is full, cover Cap the centrifuge tube, seal the bottle with a parafilm, mix it upside down, and incubate in a 37°C incubator for 48 hours to simulate the state of serum-free and hypoxic stress;

5) Centrifuge the cells incubated in the previous step at 200g/min for 5min, collect the precipitated cells, add DMEM culture medium containing 2% (v/v) FBS, mix well, and inoculate the cells into tissue culture treated common Adherent growth in cell culture dishes;

6) After the above-mentioned cells adhered to the wall, the glioma TSC medium was replaced for culture, wherein the glioma TSC medium contained 20ng/ml EGF, 10 ng/ml bFGF, 2% B27 supplement without vitamin A, 1% N2 supplement, 1×NEAA, 1×L-glutamine Neurobasal medium;

7) When the cells are congested, repeat steps 1) to 6) three times, and the obtained cells are called 3 ^rd -Muse-U251;

8) Spheroids culture: Inoculate the 3 ^rd -Muse-U251 obtained above into a tissue culture-treated cell culture dish (6-well plate contains 100-2000 cells per well), and use glioma TSC medium to inoculate single cells After a few days of low-density adherent growth, most cells formed uniform and dense clones with increased nuclear-to-cytoplasmic ratios, which were significantly different from the original U251 morphology and had typical stem cell characteristics, while very few clones maintained the original cell morphology of U251. The heterogeneous non-TSC was scraped off with a scraper needle; the overgrown 3 ^rd -Muse-U251 was digested into single cells, and the glioma TSC medium was used to suspend them into the agarose-lined cell culture dish for suspension culture. The obtained Spheroids (microspheres) are glioma stem cells, which are called U251-Muse TSCs, and the purification process of digestion into single cells and suspension culture was repeated three times to obtain smoother and more stable U251-Muse TSCs (see Figure 9 );

The U251-Muse TSC prepared in Example 3 is identified below.

(1) Identification by flow cytometry (FACS)

Using FACS to identify the tumor stem surface marker CD133 in common U251 cells (Figure 10A) and U251-Muse TSC prepared in Example 3 (Figure 10B), the results showed that more than 93% of U251-Muse TSCs had CD133 markers, About 2% of U251 cells contain CD133 (as shown in Figure 10).

(2) Low serum colony formation assay

Take 1000 common glioma cell lines U251, traditional U251 spheroids-TSC and U251-Muse TSC cells prepared in Example 3 to detect the self-renewal ability of each type of cells, and inoculate the above three types of cells into normal Concentration (10%), low concentration (2%) and very low concentration (0.5%) of serum FBS cultured for 9 days after single cell adherent culture, crystal violet staining was used to detect the colony formation of each cell.

The experimental results show that the common tumor cell U251 has good colony formation ability only in the normal concentration (10%) serum; It has good colony formation ability, but the colony formation ability is very poor under the condition of extremely low concentration (0.5%) serum; however, the U251-Muse TSC prepared in Example 3 is still under the condition of extremely low concentration (0.5%) serum. Maintaining good clone formation ability (see Figure 11), it can be seen that the quality and purity of the Muse TSC separated by the Muse TSC system of the present invention are better than those of the traditional Spheroids-TSC. the

Example 4 Isolation method of breast cancer stem cells

1) Digest adherent breast cancer cells MCF7 with 0.25% trypsin solution at 37°C for 4 minutes to single cells, terminate with medium containing serum, centrifuge at 200g/min for 5min, and collect the precipitated cells;

2) Add 0.1% type I collagenase solution (prepared from basal medium DMEM/F12 culture medium) to the collected cells above, add 1mL enzyme solution per 1× ¹⁰⁶ cells, incubate at 37°C for 6h, simulate Digestive enzyme stress state;

3) Move the above-incubated cells to 4°C and incubate for 12-16 hours to simulate low temperature and digestive enzyme stress state;

4) Wash the incubated cells with PBS buffer, mix well, centrifuge at 200g/min for 5min, discard the supernatant, wash off the enzyme solution, add DMEM/F12 culture medium to the cells precipitated in the centrifuge tube until the centrifuge tube is full , cover the cap of the centrifuge tube, seal the bottle mouth with a parafilm, mix it upside down, and incubate it in a 37°C incubator for 48 hours to simulate the state of serum-free and hypoxic stress;

5) Centrifuge the cells incubated in the previous step at 200g/min for 5min, collect the precipitated cells, add DMEM/F12 culture medium containing 2% (v/v) FBS, mix well, and inoculate the cells into tissue culture treated Adhesive growth in ordinary cell culture dishes;

6) After the above cells adhered to the wall, the breast cancer TSC medium was replaced for culture, and the breast cancer TSC medium was DMEM/F12 medium containing 10 ng/ml EGF, 20 ng/ml bFGF, 0.4% BSA, and 5 μg/ml insulin;

7) When the cells are congested, repeat steps 1) to 6) three times, and the obtained cells are called 3 ^rd -Muse-MCF7;

8) Spheroids culture: Inoculate the 3 ^rd -Muse-MCF7 obtained above into a tissue culture-treated cell culture dish (6-well plate contains 100-2000 cells per well), and use breast cancer TSC medium at a single cell low Density adherent growth, after a few days, most cells form uniform and dense clones with increased nuclear-to-cytoplasmic ratios, which are significantly different from the original MCF7 morphology and have typical stem cell characteristics, while a very small number of clones maintain the original cell morphology of MCF7. The heterogeneous non-TSC was scraped off with a scraper needle; the overgrown 3 ^rd -Muse-MCF7 was digested into single cells, and the breast cancer TSC medium was used to suspend them in a cell culture dish with agarose pad for suspension culture, and the obtained Spheroids (microspheres) are breast cancer stem cells, called MCF7-Muse TSC, and then repeated 3 times of digestion into single cells and purification of suspension culture to obtain smoother and more stable MCF7-Muse TSC (see Figure 12);

The MCF7-Muse TSC (breast tumor stem cell) prepared in Example 4 will be identified below.

Flow Cytometry (FACS) Identification

Using FACS to identify the tumor stem surface marker CD133 in common MCF7 cells (Figure 13A) and MCF7-Muse TSC prepared in Example 4 (Figure 13B), the results showed that more than 94% of MCF7-Muse TSCs had CD133 markers, About 1% of MCF7 cells contain CD133 (as shown in Figure 13).

The above experimental results show that the Muse TSC system established by the present invention can produce TSCs of various types of tumors stably, with high quality, simply and economically.

The above examples are only the preferred cases of the present invention. According to the understanding of those skilled in the art, the method of the present invention can also be widely applied to the isolation of other tumor stem cells. During the separation process, relevant conditional parameters (such as digestion time, centrifugation time, medium, digestive enzymes, etc.) can also be adjusted appropriately according to the difference of tumor cells.

Therefore, for those skilled in the art, any obvious changes and improvements within the scope of not departing from the spirit of the present invention should be regarded as a part of the present invention.

Claims (8)

1. A method for separating tumor stem cells, characterized in that: comprising the following steps: 1) After digesting the adherent tumor cells to a single cell with digestive enzyme solution 1, stop the digestion, centrifuge, and collect the precipitated cells; 2) Add digestive enzyme solution 2 to the collected cells and incubate for 0.5-10 hours; 3) Wash and centrifuge the incubated cells, collect the precipitated cells, add serum-containing cell culture medium, mix well, inoculate the cells into cell culture dishes for adherent growth; 4) After the above cells adhered to the wall, the tumor stem cell TSC medium was replaced for culture; 5) When the cells are full, repeat steps 1) to 4) 2 to 6 times, and the obtained cells are called Muse-tumor cells; 6) Spheroids culture: Inoculate the Muse-tumor cells obtained above into a cell culture dish, and use TSC medium to make the Muse-tumor cells grow single-cell and low-density adherent. When most cells form uniform and dense clones, the nucleus and cytoplasm When the ratio increases and has typical stem cell characteristics, remove a small number of heterogeneous non-tumor stem cells, digest the overgrown Muse-tumor cells into single cells, and use TSC medium to suspend to agarose-matched or low-adhesion cells Suspension culture in a petri dish, the obtained microsphere Spheroids are tumor stem cells, called Muse TSC. 2. A method for separating tumor stem cells according to claim 1, characterized in that: comprising the following steps: 1) After digesting the adherent tumor cells to a single cell with digestive enzyme solution 1, stop the digestion, centrifuge, and collect the precipitated cells; 2) Add digestive enzyme solution 2 to the collected cells and incubate at 1-37°C for 0.5-10 hours to simulate the stress state of digestive enzymes; 3) Move the above-incubated cells to 1-18°C and continue to incubate for 10-72 hours to simulate the low temperature and digestive enzyme stress state; 4) Wash the incubated cells, centrifuge, collect the precipitated cells, add serum-free or serum-less than 5% (v/v) cell culture medium, mix well, and store at 1-37°C under airtight conditions Incubate for 20-70 hours under low temperature to simulate the state of serum-free and hypoxic stress; 5) Centrifuge the cells incubated in the previous step, collect the precipitated cells, add cell culture medium containing 1-20% (v/v) FBS, mix well, inoculate cells into cell culture dishes for adherent growth; 6) After the above cells adhered to the wall, the tumor stem cell TSC medium was replaced for culture; 7) When the cells are full, repeat steps 1) to 6) 2 to 6 times, and the obtained cells are called Muse-tumor cells; 8) Spheroids culture: Inoculate the Muse-tumor cells obtained above into a cell culture dish, and use TSC medium to make the Muse-tumor cells grow single-cell and low-density adherent. When most cells form uniform and dense clones, the nucleus and cytoplasm When the ratio increases and has typical stem cell characteristics, remove a small number of heterogeneous non-tumor stem cells, digest the overgrown Muse-tumor cells into single cells, and suspend them into agarose-matched cell culture dishes using TSC medium. Suspension culture, the obtained microsphere Spheroids are tumor stem cells, called Muse TSC. 3. A method for isolating tumor stem cells according to claim 1 or 2, characterized in that: the digestive enzyme in the digestive enzyme liquid 1 is selected from one of trypsin, TrypLE, and Accutase. 4. A method for isolating tumor stem cells according to claim 1 or 2, characterized in that 0.2-15 mL of digestive enzyme solution 2 is added to every 1×10 ⁶ to 9×10 ⁶ cells in step 2). 5. A method for isolating tumor stem cells according to claim 1, 2 or 4, characterized in that: the digestive enzyme solution 2 is selected from 0.02-0.3% (w/v) type I collagenase solution, 0.02 ~0.3% (w/v) type IV collagenase solution, 0.1~3U/ml Dispase solution, 0.01~0.3% (w/v) trypsin solution, 0.01~1% (w/v) TrypLE Enzyme solution, one of 0.01-1% (w/v) Accutase enzyme solution. 6. The method for isolating tumor stem cells according to claim 1 or 2, characterized in that: all the cell culture fluids are selected from different cell basal media according to different types of tumor cells. 7. The separation method of a kind of tumor stem cells according to claim 1 or 2, characterized in that: in the Spheroids culture, the number of Muse-tumor cells inoculated into the cell culture dish is that each well of a six-well plate contains 100 ~2000 cells. 8. A method for isolating tumor stem cells according to claim 1 or 2, characterized in that: the obtained Muse TSCs are digested into single cells and cultured in suspension repeatedly at least once to obtain higher purity and smoother Stabilized microsphere Spheroids, namely Muse TSC.