CN112501121A

CN112501121A - Basic culture medium and culture method for circulating tumor cells

Info

Publication number: CN112501121A
Application number: CN202011333758.9A
Authority: CN
Inventors: 吴诗扬; 刘志明; 许嘉森; 黄洁芬; 曾杰
Original assignee: Surexam Bio Tech Co Ltd
Current assignee: Surexam Bio Tech Co Ltd
Priority date: 2020-11-24
Filing date: 2020-11-24
Publication date: 2021-03-16
Anticipated expiration: 2040-11-24
Also published as: CN112501121B

Abstract

The invention relates to a basic culture medium and a culture method for circulating tumor cells. The culture medium of the invention is added with a plurality of compound components, including inactivated Streptomyces roseochromogenes fermentation liquor. The secondary metabolite in the fermentation liquor of the streptomyces ochromorpha strain can be used as a nutrient component for cell growth to promote cell proliferation, and meanwhile, the generated bacteriostatic component can effectively inhibit bacteria in a cell culture medium, so that the use of antibiotics in the cell culture medium is reduced, the cell growth state is good under the condition of not additionally adding serum, the culture cost is reduced, the problem of possible culture difference caused by the use of different batches of serum is solved, and the application scene is economically and efficiently expanded. Meanwhile, the primary tumor cells can be subjected to in-vitro large-scale and infinite subculture, a large number of cells are provided for the development of tumor drug sensitivity tests, and the possibility is created for the establishment of a cell model.

Description

Basic culture medium and culture method for circulating tumor cells

Technical Field

The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and particularly relates to a basic culture medium and a culture method for circulating tumor cells.

Background

In 1869, the concept of Circulating Tumor Cells (CTCs) was first proposed by Ashworth, an australian doctor. In 1976 Nowell revised the definition of CTC to: derived from primary or metastatic tumors, acquire the ability to detach from the basement membrane and invade tumor cells that enter the blood vessels through the tissue matrix. CTCs are now a generic term for the types of tumor cells present in peripheral blood.

CTCs are useful for cancer diagnosis and monitoring of cancer status, and are key to tracking tumor cell metastasis. Clinical studies have demonstrated that CTCs are associated with disease progression in a variety of cancers. Therefore, the detection of the CTCs plays an important guiding role in patient prognosis judgment, curative effect evaluation and individualized treatment.

In recent years, the detection technology of CTCs is continuously developed, and due to different enrichment and detection methods, the detection method of CTCs in clinical practice is not standardized, and the main application of the detection method is limited to simple counting. Although the count of CTCs has great guiding value for early diagnosis and clinical treatment of cancer, it has many difficulties and challenges in detection. First, the number of CTCs in peripheral blood is very small (about 1-100 CTCs per ml of blood), which requires that the detection method can efficiently and accurately detect very few target cells from a large number of non-target cells. Slight deviation, large influence on the detection result and easy occurrence of contingency. Secondly, studies have shown that CTCs are highly heterogeneous in that partial or complete epithelial-mesenchymal transition of CTCs into peripheral blood has occurred, whereas current detection methods are hardly able to distinguish between subpopulations of CTCs, nor is it clear from which subpopulation CTCs having the ability to form micrometastases or metastases originate. Finally, due to shear forces in the peripheral circulation, immune system attack, and loss of cell attachment and cell-matrix junctions, most CTCs in the blood are in an apoptotic state, and only a small fraction of CTCs with highly active and metastatic potential can survive in the blood circulation and undergo distant organ metastasis. This fraction is most likely to be the actual clinically significant CTCs of therapeutic importance. Which is not known by simply counting CTCs.

Therefore, achieving efficient in vitro amplification of a very small number of CTCs would break through this bottleneck. At present, the literature reports on in vitro culture of CTCs are very limited, and the reasons may be that cells are difficult to enrich, culture conditions are inappropriate and the like, so a high-efficiency and mild CTCs enrichment method is urgently needed to be developed, and a new in vitro culture method is adopted to effectively realize in vitro rapid amplification of the CTCs, so that comprehensive molecular and functional research on the CTCs is realized. The method is more favorable for improving the reference evaluation value of the detection result and assisting in clinically obtaining a more objective detection result, so that the treatment scheme of the patient is optimized and the treatment efficiency is effectively improved.

In view of this, the invention is particularly proposed.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a basic culture medium for circulating tumor cells, which can replace serum and realize high-efficiency and rapid culture of the circulating tumor cells.

The technical scheme for achieving the purpose is as follows.

A basic culture medium for circulating tumor cells is prepared by adding a fermentation liquid of inactivated Streptomyces roseoflavus into an RPMI1640/F12 culture medium.

In some of these examples, the fermentation broth of Streptomyces roseoflavus is added in an amount of 1-20. mu.g/ml (total concentration), more preferably in a concentration range of 3-10. mu.g/ml, still more preferably 3-8. mu.g/ml.

In some of these examples, the basal medium is supplemented with 5-10ng/mL basic fiber growth factor FGF2, 5-8. mu.g/mL human recombinant insulin, 0.8-1.2mM sodium pyruvate, 8-12mg/L transferrin, 0.5-1mM N-acetylcysteine, 7-11mM nicotinamide, 0.5-0.7mg/mL taurine, 30-70U/mL penicillin and 30-70. mu.g/mL streptomycin, 3-8. mu.g/mL fermentation broth of inactivated Streptomyces roseochromogenes.

In some embodiments, the basic medium is supplemented with 8 + -1 ng/mL basic fiber growth factor FGF2, 6 + -1 μ g/mL human recombinant insulin, 1 + -0.1 mM sodium pyruvate, 11 + -1 mg/L transferrin, 0.7 + -0.05 mM N-acetylcysteine, 9mM nicotinamide, 0.6 + -0.05 mg/mL taurine, 50 + -3U/mL penicillin, 50 + -3 μ g/mL streptomycin, 5 + -1 μ g/mL inactivated S.roseochromogenes broth.

In some of these examples, the basal medium is supplemented with 8ng/mL basic fiber growth factor FGF2, 6. mu.g/mL human recombinant insulin, 1mM sodium pyruvate, 11mg/L transferrin, 0.7mM N-acetylcysteine, 9mM nicotinamide, 0.6mg/mL taurine, 50U/mL penicillin, 50. mu.g/mL streptomycin, 5. mu.g/mL fermentation broth of inactivated Streptomyces roseochromopes.

In some of these embodiments, the RPMI1640/F12 medium is F12 medium and the RPMI1640 medium is prepared at a ratio of 1: 0.8-1.2.

In some of these embodiments, the fermentation broth of the inactivated streptomyces roseoflavus is added as a lyophilized powder.

In some embodiments, the preparation of the fermentation broth lyophilized powder of streptomyces roseoflavus comprises the following steps:

selecting strains to carry out seed fermentation in a seed culture medium, and carrying out constant-temperature shaking culture at the temperature of 28 +/-1 ℃ for four days to obtain a seed solution; inoculating the obtained seed solution into sterilized ISPII culture medium at an inoculation amount of 6 + -0.5%, fermenting and culturing for 12 + -1d, centrifuging after culturing, separating fermentation broth supernatant, adding activated carbon according to a solid-to-liquid ratio of 1:9-11, and filtering with filter paper after static adsorption; and filtering with a filter membrane, and performing vacuum freeze drying to obtain the product.

Another object of the present invention is to provide a method for culturing circulating tumor cells.

The technical scheme for achieving the purpose is as follows.

1) Adding the primary tumor cells into a basic culture medium of the circulating tumor cells to prepare a primary tumor cell suspension; or adding the cell line into the basic culture medium of the circulating tumor cells, uniformly mixing, and then adding the basic culture medium of the circulating tumor cells containing serum to prepare cell line cell suspension;

2) then adding the mixture into a basic culture medium of the circulating tumor cells, culturing in an incubator with 37 +/-2 ℃, 5 +/-0.5% CO2 and saturated humidity, changing the basic culture medium of the circulating tumor cells after 2 +/-1 days, removing suspended cells, and then changing the culture solution once every 2 +/-0.5 days.

In some embodiments, the technical scheme is as follows:

1) adding the primary tumor cells into the basic culture medium of the circulating tumor cells to prepare a primary tumor cell suspension; or adding the cell line into the culture medium, mixing uniformly, and adding a basic culture medium containing the circulating tumor cells of the serum to prepare cell line cell suspension;

2) centrifuging the cell suspension to remove supernatant, mixing with Matrigel, standing, adding 500 + -10 μ L of the basic culture medium for circulating tumor cells, and adding 5 + -0.5% CO₂Culturing in 37 + -2 deg.C incubator, observing cell growth, and changing culture medium every 2 + -1 days.

In some embodiments, the technical scheme is as follows:

1) adding the primary tumor cells into the basic culture medium of the circulating tumor cells to prepare a primary tumor cell suspension; or adding the cell line into the basic culture medium of the circulating tumor cells, uniformly mixing, and adding the basic culture medium of the circulating tumor cells containing serum to prepare cell line cell suspension;

2) freezing and thawing the Matrigel into liquid, uniformly coating the Matrigel on the bottom of a pore plate, and standing the culture plate in a constant-temperature carbon dioxide incubator at the temperature of 37 +/-2 ℃ for 20 +/-2 minutes to solidify the liquid Matrigel into colloid;

3) adding the cell suspension into a pore plate which is paved with glue in advance, culturing in an incubator at 37 +/-2 ℃, 5 +/-0.5% CO2 and saturated humidity, changing the liquid after 2 +/-1 days, removing the suspended cells, and then changing the liquid once every 2 +/-1 days.

The invention also provides a complete culture medium for circulating tumor cells.

A complete culture medium for circulating tumor cells is composed of the basic culture medium for circulating tumor cells and serum added into the basic culture medium.

The basic culture medium of the circulating tumor cells can also be used for preparing a complete culture medium. The complete culture medium can be used for culturing circulating tumor cells (primary CTC) separated from a sample to be detected or directly used for culturing a tumor cell line (mature tumor cell line), and consists of the basic culture medium of the circulating tumor cells and serum, wherein the serum is supernatant obtained after the sample to be detected is subjected to coagulation centrifugation, and is filtered by a microporous filter membrane to obtain or commercialize fetal calf serum.

The basic culture medium for circulating tumor cells is not only suitable for two-dimensional cell culture, but also can be further applied to three-dimensional cell culture. When the basic culture medium for circulating tumor cells is used for culturing the tumor cells, the Streptomyces Roseoflavus (SR) fermentation liquid (freeze-dried powder) is added, secondary metabolites in the strain fermentation liquid can be used as nutrient components for cell growth to promote cell proliferation, meanwhile, the generated bacteriostatic components can effectively inhibit bacteria in a cell culture medium, the use of antibiotics in the cell culture medium is reduced, further, other proper auxiliary components are added, the cell growth state is good under the condition of not additionally adding serum, the culture cost is reduced, the problem of difference of possible culture caused by the use of different batches of serum is solved, and the application scene of the basic culture medium is economically and efficiently expanded. Meanwhile, the primary tumor cells can be subjected to in-vitro large-scale and infinite subculture, a large number of cells are provided for the development of tumor drug sensitivity tests, and the possibility is created for the establishment of a cell model.

The basic culture medium for culturing the circulating tumor cells can be used for primary culture of the CTCs, so that the CTCs can be rapidly proliferated in the basic culture medium for the circulating tumor cells, and other non-tumor cells are poor in growth state in the basic culture medium for the circulating tumor cells, so that the basic culture medium for the circulating tumor cells not only achieves the purposes of screening and impurity removal, but also can enrich the CTCs simultaneously, and provides sufficient cell samples for subsequent downstream pathology research and related molecular typing detection. Meanwhile, the basic culture medium of the circulating tumor cells can also be used for the rapid culture of tumor cell lines.

The invention solves the problem that the primary tumor cells are difficult to culture in vitro, and the cultured tumor cells can be used for screening novel antitumor drugs or detecting the sensitivity of the tumor cells to different antitumor drugs. The drug sensitivity detection experiment can improve the accuracy of the drug administration of tumor patients and provide scientific basis for clinicians to determine individualized treatment schemes and carry out individualized treatment.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1: analysis statistical chart of influence of different concentrations of streptomyces roseoflavus fermentation broth freeze-dried powder on activity of HEK293 cells in example 2;

FIG. 2: statistical analysis of cell growth in different media as in example 4;

FIG. 3: cell growth profiles in the 4 groups of media in example 4;

FIG. 4: comparative plots (100X) of the culture conditions of the cells in the experimental group 3 of example 4 on the first and seventh days;

FIG. 5: comparative plots of the culture conditions of the cells cultured in the experimental group 11 of example 4 for the first day (100X) and the seventh day (200X);

FIG. 6: comparative graphs of the culture states of the experimental groups 1-B in example 5 from 0 to 1 d;

FIG. 7: the detection result of CTC cultured in experimental group II-A in example 5 is shown by observing under a 20X lens, wherein A is a bright field image, B is a synthetic image, C is a DAPI image, and D is EpCAM-PE;

FIG. 8: example 6 comparison of cell states after three-dimensional culture using different media (100X), A being the cell state after three-dimensional culture using the medium of the present invention, B being the cell state after three-dimensional culture using the comparison medium;

FIG. 9: FIG. 400X shows the results of cell detection in example 6 after culture using the medium of the present invention, wherein A represents 2D culture and B represents 3D culture;

FIG. 10: cell detection map (400X) after culture in example 6 using comparative medium, a for 2D culture and B for 3D culture.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It will be appreciated that the experimental procedures for the following examples, where specific conditions are not indicated, are generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various reagents used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Primers and probes used in the present application can be prepared using standard techniques well known in the art.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1 preparation of Streptomyces roseoflavus fermentation broth lyophilized powder

Firstly, the source of the strain

Purchased from China center for culture collection and management of industrial microorganisms: streptomyces roseoflavus (Streptomyces roseofluscus, accession number of the strain: ACCC40123 (platform number: bio-52663), hereinafter referred to as SR for short in the laboratory), is known to be useful for inhibiting gram-positive bacteria, gram-negative bacteria and filamentous fungi.

Secondly, preparation of culture medium

1) Seed medium (g/L):

glucose 20.0g, peptone 6.0g, NaCl10.0g, pH 7.0.

2) ISP2 medium (proposed medium for international conference on streptomyces planning):

4.0g of yeast extract, 10.0g of malt extract and 4.0g of glucose, adjusting pH after dissolution and fixing the volume to 1.0L, performing steam sterilization at 115 ℃ and high pressure (1.034 multiplied by 105Pa) for 30 minutes (the glucose can be decomposed into the culture of harmful microorganisms of sugar acid at high temperature and high pressure, and the glucose can not be decomposed while performing disinfection and sterilization at 115 ℃ for 30 minutes), and storing at 4 ℃ for later use.

Thirdly, strain recovery and fermentation liquid freeze-dried powder preparation

Selecting a proper amount of bacterial strains to carry out seed fermentation in a seed culture medium, and carrying out shaking culture at a constant temperature of 28 ℃ for four days to obtain a seed solution. Inoculating the obtained seed liquid into a sterilized ISPII culture medium (solid GYM: 4g of glucose, 4g of malt extract, 10g of yeast powder, 2g of calcium carbonate and 18g of agar powder, adding water to 1000 ml, sterilizing) for liquid fermentation, performing fermentation culture for 12 days in a rotary temperature-controlled shaking table (28 ℃ and 180rpm), after the culture is finished, centrifuging for 10min at 4000rpm and 4 ℃ by using a table-type high-speed refrigerated centrifuge, separating a fermentation liquid supernatant in an aseptic environment, adding activated carbon according to a solid-to-liquid ratio of 1:10, and filtering by using filter paper after static adsorption for 3 hours. Then filtering with 0.22 μm filter membrane, and vacuum freeze drying to obtain the final product.

Example 2 Streptomyces roseoflavus fermentation broth freeze-dried powder cytotoxicity experiment

HEK293 cells (human embryonic kidney cells) were inoculated into culture flasks, and RPMI1640 medium (Gibco) containing 10% fetal bovine serum was used at 37 ℃ with 5% CO₂Culturing in the environment for 12-72 h. Taking cells in logarithmic growth phase, re-suspending the cell suspension with culture medium, and adjusting the density to be 1 × 10⁵one/mL, 100. mu.L of LPBS was added to peripheral wells of a 96-well plate using a multichannel pipette, and 100. mu.L of LPBS was added to the remaining wells at a density of 1X 10⁵Cell suspension per mL. Cells were incubated at 37 ℃ with 5% CO₂The incubator is incubated for 24 hours, and the culture medium is sucked outmu.L of culture medium containing SR fermentation broth lyophilized powder with concentration of 0. mu.g/ml (control group), 0.3125. mu.g/ml, 0.625. mu.g/ml, 1.25. mu.g/ml, 2.5. mu.g/ml, 5. mu.g/ml, 10. mu.g/ml, 15. mu.g/ml and 20. mu.g/ml is added to each well. Placing in an incubator, continuously culturing for 24h, taking out, absorbing the culture medium, adding culture medium containing 10% CCK-8 reagent into each well, continuously culturing for 4h, and measuring absorbance value of each well by using an enzyme-labeling instrument (ELX800 full-automatic enzyme-labeling instrument, Proteus instruments, Ltd.) under the condition that the excitation wavelength is 450nm, wherein only the culture solution and CCK-8 with the same amount are added as blank wells. Cell viability was calculated according to the following formula: cell survival (%) - (experimental well absorbance-blank well absorbance)/(control well absorbance-blank well absorbance) × 100%. For each concentration 6 parallel wells were set, the experiment was repeated 3 times, and the relevant experimental results were recorded and calculated and filled out in the table below.

TABLE 1

SR fermentation liquor freeze-dried powder	Multiple holes 1	Multiple holes 2	Multiple holes 3	Multiple holes 4	Multiple holes 5	Multiple holes 6	Mean value of
								0.3125μg/ml	0.912	0.909	0.907	0.923	0.916	0.914	0.9135
0.625μg/ml	0.924	0.922	0.931	0.923	0.933	0.921	0.9257
								1.25μg/ml	0.924	0.928	0.919	0.922	0.928	0.917	0.923
2.5μg/ml	0.948	0.934	0.942	0.938	0.929	0.936	0.9378
								5μg/ml	0.953	0.947	0.959	0.963	0.960	0.955	0.9562
10μg/ml	0.948	0.931	0.940	0.941	0.943	0.936	0.9398
								15μg/ml	0.901	0.923	0.908	0.897	0.915	0.911	0.9092
20μg/ml	0.904	0.901	0.913	0.905	0.908	0.903	0.9057
								Control group (0. mu.g/ml)	0.921	0.937	0.930	0.923	0.932	0.925	0.928
Blank group	0.211	0.209	0.233	0.217	0.223	0.231	0.2207

The survival rate of each group of cells was calculated from the above average value, and the results of repeating three cell plates were as follows:

TABLE 2

SR fermentation liquor freeze-dried powder	Cell viability	1	Cell viability 2	Cell viability 3	Mean value of
						0.3125μg/ml	97.9％	96.7％	97.1％	97.2％
0.625μg/ml	99.7％	98.8％	99.2％	99.2％
					1.25μg/ml	99.3％	99.5％	99.6％	99.5％
2.5μg/ml	102.1％	101.6％	102.4％	102％
					5μg/ml	104.0％	103.5％	104.2％	103.9％
10μg/ml	101.7％	100.8％	101.2％	101.2％
					15μg/ml	97.3％	96.7％	96.2％	96.7％
20μg/ml	96.8％	95.8％	95.9％	96.2％

The results are statistically shown in the table above and in FIG. 1.

The results show that the culture medium containing the streptomyces roseochromosus fermentation broth freeze-dried powder within the use concentration range has low cytotoxicity, the cell survival rate is more than 95 percent, the culture medium has no inhibition effect on cell growth, and the cell proliferation is obvious under partial concentration. However, in the above-mentioned range, the optimum concentration range is 3 to 10. mu.g/ml in view of comprehensive utilization efficiency and cost.

EXAMPLE 3 kit composition and Using procedures

The kit mainly comprises a circulating tumor cell basic culture medium which specifically comprises the following components in concentration: RPMI1640/F12 medium, 8ng/mL basic fiber growth factor FGF2, 6 μ g/mL human recombinant insulin, 1mM sodium pyruvate (antioxidant), 11mg/L transferrin, 0.7mM N-acetylcysteine, 9mM nicotinamide (vitamin B3 derivative), 0.6mg/mL taurine, 50U/mL penicillin, 50 μ g/mL streptomycin, 5 μ g/mL Streptomyces Roseochromogenes (SR) fermentation broth (lyophilized powder); wherein the RPMI1640/F12 medium was F12 medium (Gibco, cat # 11765062) and RPMI1640 medium (Gibco, cat # A1049101) prepared according to 1: 1.

Wherein the basic culture medium can also be used for preparing a complete culture medium. The complete medium may be composed of a basal medium and serum, wherein the serum may be whole blood obtained by coagulation centrifugation, or commercial fetal bovine serum.

The invention can be applied to primary culture or cell line culture, and the culture objects are respectively obtained according to the following steps:

primary tumor cells:

(1) taking 5ml of a blood sample to be detected, adding 15ml of circulating tumor cell enrichment preservation solution, and fully and uniformly mixing the two;

(2) standing at 4 deg.C for 20min, centrifuging at 600g for 5min, and removing supernatant.

(3) Add 4ml PBS and vortex and mix well.

(4) And (3) filtering a sample: transferring the liquid in the sample storage tube into a filter, and opening a vacuum pump to pump out the liquid; 4ml PBS was added to the tube, and the tube wall was washed and the liquid was filtered off with suction.

(5) And transferring the filter membrane into a centrifuge tube, adding 3 +/-2 mL (adaptively adding according to the specification of a culture plate or a culture bottle used for subsequent culture) of the circulating tumor cell basic culture medium, and gently blowing and beating to ensure that the CTCs on the filter membrane fall into the culture medium to form cell suspension.

Tumor cell line cells:

(1) placing the glass beaker filled with clean distilled water in a constant-temperature water bath kettle, heating at the constant temperature of 37 ℃ for 20 minutes, taking out the corresponding cell freezing tube from a liquid nitrogen tank, quickly placing the cell freezing tube in the glass beaker in the constant-temperature water bath, shaking for accelerating melting, taking out the cell freezing tube after the solid in the freezing tube is melted to be more than 2/3, wiping the tube wall with absorbent cotton, then sterilizing the whole tube with 75% alcohol, and placing the tube on a clean workbench for later use.

(2) And (3) sucking the thawed cell suspension in the cryopreservation tube by using a pipette gun, adding the cell suspension into a centrifuge tube containing 4.5 +/-2 mL of RPMI1640 culture medium, and gently and uniformly stirring by blowing.

(3) Centrifuging at 1000rpm for 5min at room temperature, discarding supernatant, adding the basic culture medium containing 10% fetal calf serum for circulating tumor cells, and resuspending cells to obtain cell suspension. Inoculating to a culture flask, 37 deg.C, 5% CO₂And culturing in an incubator with saturated humidity, changing the culture solution after 2-3 days, removing suspended cells, and then changing the culture solution once every 2 days.

The invention can be simultaneously applied to two-dimensional cell culture or three-dimensional cell culture,

one-dimensional cell culture:

adding the cell suspension into a culture flask with the circulating tumor cell culture medium at 37 deg.C and 5% CO₂And culturing in an incubator with saturated humidity, changing the culture solution after 2-3 days, removing suspended cells, and then changing the culture solution once every 2 days.

Two, three-dimensional cell culture (gel culture):

1. spreading glue

The Matrigel was placed in a refrigerator at 4 ℃ overnight and after about 12 hours, the Matrigel was observed to change from a solid state to a liquid state. Sucking 60-100 mu L of liquid Matrigel by using a suction head pre-cooled on ice in advance, gently and quickly and uniformly smearing the liquid Matrigel on the bottom of a pore plate to ensure that the thickness is uniform and bubbles are not generated, and then placing the culture plate in a constant-temperature carbon dioxide incubator at 37 ℃ for standing for 20 minutes to solidify the Matrigel matrix gel into colloid.

2. Culturing

The cell suspension was added to a well plate previously coated with gel, cultured in an incubator at 37 ℃ with 5% CO2 and saturated humidity, and the medium (basal medium described in example 3) was changed after 2 to 3 days, and suspended cells were removed, and then changed every 2 days.

Thirdly, three-dimensional cell culture (in-gel culture):

the cell suspension was centrifuged to remove the supernatant, concentrated to an appropriate concentration, and 40. mu.L of matrigel was added to the cells obtained by enrichment and the cells were suspended in the matrigel. The matrigel containing cells was dropped on the center of a 24-well plate, placed in a cell incubator at 37 ℃ for 15 minutes, 500. mu.L of the circulating tumor cell basal medium described in example 3 was added, and cultured in an incubator at 37 ℃ with 5% CO2, and the growth of the cells was observed, and the medium was changed every 2 to 3 days.

EXAMPLE 4 cell growth Curve (Medium screening optimization)

After the NCl-H1975 cell line is recovered conventionally, the cell line is completely cultured in RPMI1640 containing 10% fetal calf serum and placed at 37 ℃ under 5% CO₂And culturing in an incubator with relative humidity of 90%. Taking the cell suspension in logarithmic growth phase, re-suspending the cell suspension by using the circulating tumor cell basic culture medium, and adjusting the density to be 2.5 multiplied by 10⁴Per mL, using multiple passesPipettors added 100. mu.L LPBS to peripheral wells of a 96-well plate and 100. mu.L of cell suspension to the remaining wells. Cells were incubated at 37 ℃ with 5% CO₂The incubator (2) was incubated for 24h, the medium was aspirated, and 100. mu.L of 1-10 groups of medium as shown in the following table was added to each well. Duplicate wells were set for each group as a parallel control. After the cells of the groups are cultured for 1 to 7 days at the same time, taking out the corresponding culture plate respectively for detection as follows: the medium was aspirated off, carefully rinsed 1-2 times with PBS, 100. mu.L of LTCA (trichloroacetic acid) was added to each well, and fixed at 4 ℃ for 1 hour; discarding TCA, washing with deionized water for 2-5 times, and drying. Add 100. mu.L of 0.2% SRB (sulforhodamine B) dye solution into each well, dye for 30 minutes at room temperature, discard the dye solution, wash away the unbound dye solution with 1% acetic acid, and dry. 200 mul 10mmol/L unbuffered Tris lye with pH10.5 is added into each hole, the shaking table is placed to shake for 20 minutes at low speed, the OD value of each hole is measured under the A490 wavelength of the microplate reader, the record and the analysis are carried out, and a growth curve is drawn.

In order to further confirm the influence degree of the streptomyces roseoflavus fermentation liquid freeze-dried powder in the circulating tumor cell culture medium formula on cell culture, the following experiment is set. Wherein the antibiotic is a commercially available double antibody (Gibco), cat #: 10378016.

TABLE 3

TABLE 4 OD values (mean) of the respective experimental groups

Drawing a corresponding histogram according to the experimental data in the table, and analyzing the histogram to show that the influence of the cell growth time of the experimental groups 1-2, 9-10 and 11 on the cell growth shows a trend of increasing and then slightly decreasing along with the prolonging of the cell growth time in a proper culture time, the cell growth of the experimental groups 3-8 increases along with the increasing of the time in the statistical time, wherein the OD value of each experimental group within 1-5d increases rapidly, and the cell growth is faster; the increase in OD was weak at 5 to 7 days. In the 11 th group, the added freeze-dried thallus powder of the inactivated streptomyces roseoflavus instead of the fermentation liquid of the streptomyces roseoflavus has the effect similar to that of the experimental group 10, although the effect is slightly better than that of the experimental group 10, the effect is still insufficient for replacing fetal calf serum in a culture medium, and after the cells are cultured for 6 days, the number of adherent cells is reduced, vacuoles and black spots can appear in the cells, and the cells are flat, irregular in shape and obviously aged (see fig. 5).

By comprehensively analyzing the data of the experimental groups 3-8, it can be seen that the growth state of the experimental group 5 is optimal, the growth state of the experimental group 3 is slightly inferior, but the concentration of the SR fermentation broth lyophilized powder used in the culture medium of the experimental group 3 is 5 mug/ml, and the concentration used in the experimental group 5 is 20 mug/ml, so that the culture medium formula of the experimental group 3 is optimized to save resources while obtaining excellent effects.

Aiming at the

experimental groups

1, 3 and 9, the growth curves of the experimental groups are further compared to show that the culture effect of the experimental group 3 is almost consistent with that of the experimental group 1 (containing serum and a double antibody group) and is slightly superior to that of the experimental group 1, which shows that the culture medium can effectively replace fetal calf serum in the traditional culture medium, thereby further increasing the practicability of the culture medium and reducing the problems of uncertainty, instability and the like caused by adding different batches of serum in cell culture.

NCl-H1975 cells can normally adhere to the wall after being cultured for 1d in the experimental group 9, the experimental group 11 and the experimental group 3, and when the cells are cultured for 7d, the cells of the experimental group 9 and the experimental group 11 adhere to the wall in a small number, are flat and irregular in shape and are aged obviously; the number of adherent cells in the experimental group 3 is obviously more, vacuoles and black spots rarely occur in the cells, and the growth state of most cells is good, which is shown in figure 4. The freeze-dried powder of the fermentation liquid of the streptomyces roseoflavus is better used as the in-vitro culture environment of the tumor cells when the concentration is 5 mu g/ml.

The screening steps aiming at the mixture ratio of other components in the circulating tumor cell culture medium are consistent with those of the circulating tumor cell culture medium, and specific data are omitted. The components at the obtained optimum use concentrations were further subjected to the following combinations of experimental groups, only a selected part of which is described below), thereby studying the optimum formulation of the medium of the present invention.

TABLE 5

TABLE 6 OD values (mean number) of the respective experimental groups

According to the analysis of the recorded experimental results in the table, the OD values of the combination of the culture medium containing the SR and the culture of the conventional culture medium (RPMI1640/F12 culture medium, 5% FBS and 1% antibiotic) are all larger than the detection values under the same conditions, namely, the culture effects of the combination of the culture medium containing the SR and the culture of the conventional culture medium are better than those of the culture of the conventional culture medium, further research on experimental groups 2-5 shows that growth promoting factors are added in the experimental group 2, auxiliary components are added in the experimental group 3 compared with the experimental group 2, double antibodies are added in the experimental group 4 compared with the

experimental groups

2 and 3, and the OD value of the experimental group 4 is increased to the maximum, which shows that the added components can promote cell growth and propagation. The fermentation broth of Streptomyces Roseoflavus (SR) was added to experimental groups 2-4, while the inactivated SR was added to experimental group 5, and the culture effect was not good due to the lack of secondary metabolites only in the inactivated SR, so that the OD value of experimental group 5 was lower than that of other experimental groups. In general, the culture of cells using the culture medium of experiment 4 group was most effective.

EXAMPLE 5 culture of CTCs

Blood samples (I-III) from breast cancer patients (5 ml of each blood sample A-C in 3 tubes) were taken and subjected to extraction of CTCs as described in example 3 above, and the cell suspensions obtained by the extraction were counted and inoculated on average into 24-well plates containing the same amount of the culture medium of the present invention and the comparative example medium described below for conventional two-dimensional cell culture, i.e., the culture conditions were identical except for the culture medium. The 3 groups of cells were cultured simultaneously for 7 days, counted and recorded daily. After 7 days, the test kit and the test procedures are correspondingly tested according to the test kit and the test procedures disclosed in the patent document (patent number: 2016100442557) previously published by the applicant, and the relevant experimental data are photographed and recorded.

And (3) comparing culture conditions:

group a samples used medium: the invention relates to a circulating tumor cell basic culture medium;

the circulating tumor cell culture medium is composed of the following components in concentration: RPMI1640/F12 medium, 8ng/mL basic fiber growth factor FGF2, 6 μ g/mL human recombinant insulin, 1mM sodium pyruvate, 11mg/L transferrin, 0.7mM N-acetylcysteine, 9mM nicotinamide, 0.6mg/mL taurine, 50U/mL penicillin, 50 μ g/mL streptomycin, 5 μ g/mL Streptomyces roseochromosa (fermentation broth freeze-dried powder).

Group B samples used medium: RPMI1640/F12 medium, 5% FBS, 1% antibiotic;

group C samples used media: RPMI1640/F12 medium, 8ng/mL basic fiber growth factor FGF2, 6 μ g/mL human recombinant insulin, 1mM sodium pyruvate (antioxidant), 11mg/L transferrin, 0.7mM N-acetylcysteine, 9mM nicotinamide (vitamin B3 derivative), 0.6mg/mL taurine, 1% antibiotic.

TABLE 7 statistical Table of the culture of CTCs in samples I-III in media A-C

Since the number of CTCs isolated from a sample is rare, the ratio of cell growth substances dispersed outside cells is such that the concentration of the substances in the cells is lower than the minimum limit, and at this time, the cells cannot proliferate any more, the pH in the culture solution becomes high (alkalinity is increased), cell growth is inhibited, cells become round and cannot adhere to the wall, and even cell death is caused. Therefore, group I-B cells appeared, and 0-1d of the cells grew as shown in the figure, and the number of the cells gradually decreased to 0 with the increase of the culture time after the cells were normally inoculated and attached, thereby causing the phenomenon that the cells finally die completely.

Compared with the culture medium B, C, the CTCs of the sample groups I-III cultured by adopting the basic culture medium (namely the culture medium A) have obvious advantages in cell proliferation quantity and speed, wherein the culture medium C is the culture medium A without the addition of the SR fermentation liquid freeze-dried powder, although the growing CTCs have no phenomenon of cell reduction, the cell proliferation speed is obviously slower than that of the group A, and the cells obtained after 7 days of culture are less than that of the group A.

And (3) detecting each group of cells cultured for 7d by referring to the CTCs related detection kit and the detection steps in the prior invention of the inventor, wherein the CTCs of the group A culture medium culture sample II are detected as shown in figure 7, which shows that the separated CTCs are all epithelial types, and the CTCs obtained after culture are also all epithelial types. The other sample detection steps and methods are the same.

Example 62D vs 3D cell culture

The basic culture medium comprises the following components in concentration: RPMI1640/F12 medium, 8ng/mL basic fiber growth factor FGF2, 6 μ g/mL human recombinant insulin, 1mM sodium pyruvate, 11mg/L transferrin, 0.7mM N-acetylcysteine, 9mM nicotinamide, 0.6mg/mL taurine, 50U/mL penicillin, 50 μ g/mL streptomycin, 5 μ g/mL Streptomyces roseochromosa (fermentation broth freeze-dried powder).

NCl-H1975 cell strain is taken, resuscitated and cultured and then counted, experimental components are divided into a 3D group and a 2D group, cells adopt the basic culture medium and the conventional culture medium (namely a control, RPMI1640/F12 culture medium, 5% FBS and 1% antibiotic) respectively, and the cells are placed at the temperature of 37 ℃ and 5% CO₂And culturing in an incubator with relative humidity of 90%. Taking the cell suspension in logarithmic growth phase at 4X 10³Inoculating 200 μ l of each cell/well into a common 96-well plate, discarding the old culture medium after the expiration, washing with PBS 2 times, adding 0.25% pancreatin for digestion, and keeping the temperature at 1-2 deg.CAnd after minutes, observing the morphological change of the cells under a microscope, removing trypsin when the cell gaps are enlarged and the adherent cell protrusions are contracted, adding a culture medium to terminate the digestion, blowing and resuspending the cells for passage, and recovering the cells to obtain a 2D culture group. The same concentration of 200. mu.l was added to a Matrigel-plated 96-well plate (three-dimensional culture method II in example 3), and after the expiration, cells were collected by trypsinization, thus obtaining a 3D culture group. Each set was set with 3 replicate wells as a parallel control. The cells were taken and examined according to the detection kit and detection procedure described in the earlier patent document (patent No. CN2015109198154) by the inventor, and the relevant experimental data were photographed and recorded.

The results are shown in fig. 8, and it can be seen by analysis that the lung adenocarcinoma cell three-dimensional culture using the culture medium of the present invention has a significantly higher cell proliferation rate, more cell spheroids and a better cell growth state than the comparative culture medium.

As shown in the results of FIGS. 9-10, it can be seen from the analysis that after lung adenocarcinoma cells cultured by different culture methods using the basic culture medium of the present invention are subjected to corresponding cell detection, fluorescence spots in the probe detection results of the related gene markers are uniformly dispersed, the three kinds of stained fluorescence spots can be accurately read, and the results can be easily visually interpreted, and the experimental results can be normally detected. When the related cells cultured by adopting the contrast medium are detected by adopting the same method, the detection result of the two-dimensional cultured cells is normal, but the cells cultured by adopting the contrast medium in the three-dimensional culture mode do not obtain a good detection result, and the analysis of the generation reason of the detection result is probably because the culture environment is not adaptive, so that partial cell genes are abnormal, and the phenomenon that partial probe detection signals are obviously enhanced/weakened is generated.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A basic culture medium for circulating tumor cells is characterized in that the basic culture medium is RPMI1640/F12 culture medium added with fermentation liquor of inactivated Streptomyces roseochromogenes.

2. The basal medium for circulating tumor cells according to claim 1, wherein the amount of the fermentation broth of Streptomyces roseoflavus added is 1-20 μ g/ml, preferably 3-10 μ g/ml.

3. The basal medium for circulating tumor cells of claim 1, wherein the basal medium contains basic fiber growth factor FGF2 of 5-10ng/mL, human recombinant insulin of 5-8 μ g/mL, sodium pyruvate of 0.8-1.2mM, transferrin of 8-12mg/L, N-acetylcysteine of 0.5-1mM, nicotinamide of 7-11mM, taurine of 0.5-0.7mg/mL, penicillin of 30-70U/mL, streptomycin of 30-70 μ g/mL, and fermentation broth of inactivated Streptomyces roseochromosa of 3-8 μ g/mL.

4. The basal medium for circulating tumor cells of claim 3, wherein the basal medium contains basic fiber growth factor FGF2 of 8 ± 1ng/mL, human recombinant insulin of 6 ± 1 μ g/mL, sodium pyruvate of 1 ± 0.1mM, transferrin of 11 ± 1mg/L, N-acetylcysteine of 0.7 ± 0.05mM, nicotinamide of 9mM, taurine of 0.6 ± 0.05mg/mL, penicillin of 50 ± 3U/mL, streptomycin of 50 ± 3 μ g/mL, and fermentation broth of inactivated Streptomyces roseochromogenes of 5 ± 1 μ g/mL.

5. The basal medium for circulating tumor cells of claim 4, wherein 8ng/mL basic fiber growth factor FGF2, 6 μ g/mL human recombinant insulin, 1mM sodium pyruvate, 11mg/L transferrin, 0.7mM N-acetylcysteine, 9mM nicotinamide, 0.6mg/mL taurine, 50U/mL penicillin, 50 μ g/mL streptomycin, and 5 μ g/mL inactivated fermentation broth of Streptomyces roseochromosa are added to the basal medium.

6. The basal medium of circulating tumor cells according to any one of claims 1 to 5, wherein the RPMI1640/F12 medium is F12 medium and the RPMI1640 medium is prepared at a ratio of 1: 0.8-1.2.

7. The basal medium for circulating tumor cells according to any one of claims 1 to 5, wherein the fermentation broth of the inactivated Streptomyces roseochromogenes is added in the form of lyophilized powder.

8. The basal medium for circulating tumor cells of claim 7, wherein the preparation of the lyophilized powder of fermentation broth of Streptomyces lavendulae comprises the following steps:

9. A two-dimensional culture method of circulating tumor cells is characterized by comprising the following steps:

1) adding primary tumor cells to the basic culture medium of the circulating tumor cells according to any one of claims 1 to 8 to prepare a primary tumor cell suspension; or adding the tumor cell line into the basic culture medium of the circulating tumor cells according to any one of claims 1 to 8, uniformly mixing, and adding the basic culture medium of the circulating tumor cells containing serum to prepare a tumor cell line cell suspension;

2) then added to the basal medium of circulating tumor cells of any one of claims 1-8 at 37 ± 2 ℃ and 5 ± 0.5% CO₂And culturing in an incubator with saturated humidity, changing the basic culture medium of the circulating tumor cells after 2 +/-1 days, removing suspended cells, and then changing the culture solution once every 2 +/-0.5 days.

10. A three-dimensional culture method of circulating tumor cells is characterized by comprising the following steps:

1) adding primary tumor cells to the basic culture medium of the circulating tumor cells according to any one of claims 1 to 8 to prepare a primary tumor cell suspension; or adding the tumor cell line into the culture medium of the circulating tumor cells according to any one of claims 1 to 8, uniformly mixing, and adding a basal culture medium containing the circulating tumor cells of serum to prepare a cell line cell suspension;

2) centrifuging the cell suspension to remove supernatant, mixing with Matrigel, standing, adding 500 + -10 μ L of the basic culture medium for circulating tumor cells of claims 1-8, and adding 5 + -0.5% CO₂Culturing in an incubator at 37 +/-2 ℃, observing the growth condition of the cells, and replacing the culture medium every 2 +/-1 days;

or comprises the following steps:

1) adding primary tumor cells to the basic culture medium of the circulating tumor cells according to any one of claims 1 to 8 to prepare a primary tumor cell suspension; or adding the tumor cell line into the basic culture medium of the circulating tumor cells according to any one of claims 1 to 8, uniformly mixing, and adding the basic culture medium of the circulating tumor cells containing serum to prepare cell line cell suspension;

3) adding the cell suspension into a pore plate which is pre-coated with glue, and keeping the temperature at 37 +/-2 ℃ and 5 +/-0.5 percent of CO₂And culturing in an incubator with saturated humidity, changing the culture solution after 2 +/-1 days, removing suspended cells, and then changing the culture solution once every 2 +/-1 days.