CN112608899A - Application of serum-free medium in culturing cancer tissue-originated spheroids - Google Patents

Application of serum-free medium in culturing cancer tissue-originated spheroids Download PDF

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CN112608899A
CN112608899A CN202011324214.6A CN202011324214A CN112608899A CN 112608899 A CN112608899 A CN 112608899A CN 202011324214 A CN202011324214 A CN 202011324214A CN 112608899 A CN112608899 A CN 112608899A
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万勇
徐中华
何志华
李明
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Guangzhou Darui Biotechnology Co ltd
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Abstract

The invention discloses a serum-free culture medium which comprises a basal culture medium, serum albumin, a ROCK inhibitor, B27, Heregulin, Activin A, EGF, bFGF, nicotinamide, NEAA, Trace Elements A, Trace Elements B, Trace Elements C and 2-mercaptoethanol. The serum-free culture medium is utilized to establish a convenient, rapid and stable culture method of cancer tissue origin spheroids, the culture medium composition and preparation method in the original culture method are optimized, a stable and controllable serum-free culture scheme is provided, the problems of insufficient sample amount and limited proliferation effect of the primary tumor cells in the existing three-dimensional culture scheme are solved, and model support is provided for preparing a CTOS biological sample bank and carrying out personalized drug evaluation and screening on cancer patients.

Description

Application of serum-free medium in culturing cancer tissue-originated spheroids
Technical Field
The invention relates to the technical field of cell culture, in particular to application of a serum-free culture medium in culturing cancer tissue-originated spheroids.
Background
Malignant tumors are common diseases threatening human health, are the first of the three main causes of death of human diseases, and account for one fourth of all causes of death of the diseases. With the development of economy and the improvement of living standard, the incidence of tumors is on the rise year by year, and the incidence and death of tumors in the whole world are estimated to reach 2640 ten thousand and 1700 ten thousand respectively by 2030. The treatment of tumors mainly depends on the technical means of surgical operation, radiotherapy and chemotherapy, targeted therapy, immunotherapy and the like. Under the large background of the rapid development of precise medical technology in the world, the development of new anticancer drugs is increasingly important, and more new anticancer drugs are developed from laboratory research to clinical application. On one hand, we search for drug targets through high-throughput sequencing technology, and on the other hand, tumor drug models are also needed to verify the effectiveness of drugs. At present, common tumor models mainly comprise commercialized cell lines, primary tumor cell models, animal models and the like, and the application of the models accelerates the development process of new anticancer drugs. Particularly, the rapid development of the three-dimensional cell culture technology in recent years is expected to be applied to preclinical research of medicines and provide a basis for a clinician to formulate a personalized diagnosis and treatment scheme.
The simplest three-dimensional culture model is a spherical collection of cells, called spheroids. Although commercial cell lines in three-dimensional culture are widely used, the original tumor characteristics lost in established cell lines may not be fully restored by forced three-dimensional structure. Animal models that enable the assessment of the efficacy of a drug candidate in a microenvironment relatively close to that of the human body, consisting of stroma and host cells (including fibroblasts, endothelial cells, blood cells and immune cells), enable pharmacodynamic and pharmacokinetic assessments. The human-derived tumor xenograft (PDX) model refers to a xenograft model in which a patient's fresh tumor tissue or tumor cells are transplanted into an immunodeficient mouse in situ or ectopically, and grow depending on the environment provided by the mouse. The model can directly simulate the natural growth process of the tumor in the human body, and the original biological characteristics of the tumor are kept to the maximum extent. The nodulation time of the PDX model is generally 2-4 months, and the modeling success rate is 23% -75%. Many studies show that the PDX model can be used as a substitute model of a specific patient for screening anti-cancer drugs and guiding the personalized treatment of tumors. Compared with a two-dimensional cultured cell line, the tumor cells in the DX can better retain the characteristics of original tumor cells and are closer to the real situation of cells in vivo. However, animal models involve problems such as animal individual difference and immune rejection, and have the disadvantages of low and unstable tumor formation rate, long modeling time, high price, difficulty in establishing a high-throughput platform, and the like, and still need a lot of scientific research to solve.
Cancer tissue-originated spheroids (CTOS) are cell spheres consisting of primary tumor cells, serve as a three-dimensional culture model of the primary tumor cells, have the advantages of cell biological characteristics close to those of in-vivo primary tissues, high culture success rate, storage capability and the like, and are ideal in-vitro drug detection models. The Kondo J team published the CTOS preparation method (KONDO, J umpei, et al. proceedings of the National Academy of Sciences,2011,108.15: 6235-. Under milder conditions, the tumor tissue is incompletely digested into cell clusters, the resulting cell clusters and cells pass through a mesh filter, and the cell clusters captured by the filter are further cultured. Further culturing in embryonic stem cell culture medium, these tissue-digested primary cells can rapidly form spheroids within 24 hours. Because the anoikis is inhibited, the number of cell death is less, and the culture success rate is greatly improved. Compared with an animal model, the method has the advantages of short culture period, low cost and higher success rate, and provides possibility for clinical-grade tumor drug sensitivity test application.
However, the existing method still has certain defects, such as large cell loss amount in the preparation process of CTOS; the selection of the CTOS mainly depends on manual operation, so that the time and labor are wasted, and the flux is low; the CTOS culture has limited proliferation effect and slow proliferation, and has great limitation on clinical application of limited samples collected by tumor tissues.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an application of a serum-free culture medium in culturing cancer tissue origin spheroids. The invention is improved on the basis of the original method, optimizes the formula of the culture medium and is more beneficial to the proliferation of tumor cells; after single cells are filtered and removed and adjusted to be cultured by CTOS, the method is favorable for obtaining cell spheroid sample bodies as much as possible; when the CTOS is cultured, the CTOS is paved in a culture container coated with a collagen gel solution or directly and uniformly mixed in the collagen gel solution, the CTOS does not need to be selected one by one, and the operation is quick and convenient.
The first purpose of the invention is to provide a serum-free culture medium.
The second purpose of the invention is to provide the application of any one of the serum-free culture mediums in the culture of spheroids originated from cancer tissues.
It is a third object of the present invention to provide a method for culturing spheroids of cancer tissue origin.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention provides a convenient, rapid and stable culture method for amplifying cancer tissue origin spheroids, which solves the problems of insufficient sample amount and limited proliferation effect of primary tumor cells in the prior art. The method adopts serum-free culture medium, and the culture condition is stable and controllable. The CTOS cultured by the method can be cultured for a long time for passage, has stable genotype, can be frozen and can be continuously and stably cultured after recovery. The method can expand the number of primary tumor cells, establish a specific cell bank belonging to a patient, and provide reliable technical support for individualized treatment of clinical patients. In addition, various tumor models can be obtained by expanding the CTOS biological sample library, and the CTOS biological sample library can be widely applied to the aspects of tumor basic research, tumor individualized treatment, antitumor drug research and development and the like, so that the development of a cancer diagnosis and treatment technology is promoted.
The invention claims a serum-free culture medium, which comprises a basal culture medium, serum albumin, a ROCK inhibitor, B27, Heregulin, Activin A, EGF, bFGF, nicotinamide, NEAA, Trace Elements A, Trace Elements B, Trace Elements C and 2-mercaptoethanol.
Trace Elements A is Mediatech 99-182-CI 1000 ×;
trace Elements B is Mediatech 99-176-CI 1000 ×;
trace Elements C is Mediatech 99-175-CI 1000 ×.
Preferably, the Serum Albumin (SA) is one or a mixture of two of Bovine Serum Albumin (BSA) or Human Serum Albumin (HSA).
More preferably, the Serum Albumin (SA) is Bovine Serum Albumin (BSA), and the concentration of Bovine Serum Albumin (BSA) in the medium is 0.1% to 10% (w/v).
Further preferably, the concentration of Bovine Serum Albumin (BSA) in the medium is 1.55% (w/v).
Preferably, the ROCK inhibitor is one or a mixture of more of Y27632, thiazovivin, Fasudil (HA-1077), GSK429286A, GSK429286A, RKI-1447, Azaindole 1 (TC-S7001), GSK269962, Netarsudil (AR-13324), Y-39983, ZINC00881524, Ripasudil (K-115) hydrochloride and Hydroxyfasuldil (HA-1100).
More preferably, the ROCK inhibitor is Y27632, and the concentration of Y27632 in the culture medium is 1-50 mu M.
Further preferably, the concentration of Y27632 in the medium is 10. mu.M.
Preferably, the basal medium is one of DMEM/F12, Advanced DMEM/F-12, RPMI 1640, DMEM, or F-12.
The basal medium provides standard inorganic salts such as zinc, iron, magnesium, calcium and potassium, as well as vitamins, glucose, buffer systems and key amino acids.
Most preferably, the basal medium is DMEM/F12.
Preferably, the concentration of nicotinamide in the culture medium is 1-100 mM; the concentration of NEAA is 0.1-10% (v/v); the concentration of Trace Elements A is 0.01-1% (v/v); the concentration of Trace Elements B is 0.01-1% (v/v); the concentration of Trace Elements C is 0.01-1% (v/v); the concentration of the 2-mercaptoethanol is 0.01-1 mM; the concentration of B27 in the culture medium is 0.1-10% (v/v); the concentration of Heregulin is 1-100 ng/ml; the concentration of Activin A is 1-100 ng/ml; the concentration of the EGF is 1-200 ng/ml; the concentration of bFGF is 1-100 ng/ml.
More preferably, the concentration of nicotinamide in the medium is 10 mM.
More preferably, the amount of NEAA in the medium is 1X, i.e.the concentration of NEAA (100X) in the medium is 1% (v/v).
More preferably, the amount of Trace Elements A in the medium is 1X, i.e., the concentration of Trace Elements A (1000X) in the medium is 0.1% (v/v).
More preferably, the amount of Trace Elements B in the medium is 1X, i.e., the concentration of Trace Elements B (1000X) in the medium is 0.1% (v/v).
More preferably, the amount of Trace Elements C in the medium is 1X, i.e., the concentration of Trace Elements C (1000X) in the medium is 0.1% (v/v).
More preferably, the concentration of 2-mercaptoethanol in the culture medium is 0.1 mM.
More preferably, the amount of B27 in the medium is 1 x, i.e. the concentration of B27(50 x) in the medium is 2% (v/v).
More preferably, the concentration of Heregulin in said medium is 10 ng/ml.
More preferably, the concentration of Activin A in the medium is 10 ng/ml.
More preferably, the concentration of EGF in the medium is 50 ng/ml.
More preferably, the concentration of bFGF in the medium is 8 ng/ml.
Preferably, the medium further contains Gastrin I.
Preferably, the concentration of Gastrin I in the culture medium is 1-100 nM.
More preferably, the concentration of Gastrin I in the medium is 10 nM.
The serum-free culture medium is used for gastric cancer primary cells, and Gastrin I is added into the culture medium.
Preferably, the medium further comprises an antibiotic.
More preferably, the antibiotic is one or more of Primocin, amphotericin B, ampicillin, penicillin-G, kanamycin, gentamycin, erythromycin, MRA, neomycin, nystatin, polymyxin B, streptomycin sulfate, tetracycline, tylosin and the like.
More preferably, the antibiotic is Primocin.
Further preferably, the concentration of Primocin is 10 to 500. mu.g/ml.
Even more preferably, the concentration of Primocin is 100. mu.g/ml.
The application of any of the above serum-free culture media in the culture of spheroids derived from cancer tissues also belongs to the protection scope of the present invention.
Therefore, the invention also claims a method for culturing spheroids originated from cancer tissues, which comprises the steps of removing tissues which are not completely digested after the digestion treatment of tumor tissues, collecting cell suspension, centrifuging and discarding supernatant, adding any serum-free culture medium into cell precipitates, mixing uniformly, transferring the cell precipitates into a culture container coated with collagen gel solution, or mixing the cell precipitates and the collagen gel solution uniformly and dripping the mixture into the culture container for culture.
The collagen gel solution consists of: fraction 1, 3mg/ml type I collagen solution, fraction 2, 10 XF-12 medium, and fraction 3, NaOH, NaHCO3And mixed liquor consisting of HEPES;
wherein, the component 1: and (2) component: component 3 ═ 8:1:1 (v: v: v);
NaOH、NaHCO3and 50mM NaOH and NaHCO in mixed solution consisting of HEPES3 260mM、HEPES 200mM。
Preferably, the cell suspension is collected by filtering through a nylon filter with a pore size of 300 μm to remove undigested tissue.
Preferably, 5% CO at 37 ℃2Culturing under the condition.
Preferably, the tumor cell is a solid tumor cell of gastric cancer, colon cancer, breast cancer, lung cancer, ovarian cancer and cervical cancer.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a serum-free culture medium, further establishes a convenient, rapid and stable culture method of cancer tissue origin spheroids, optimizes the culture medium composition and preparation method in the original culture method, provides a stable and controllable serum-free culture scheme, solves the problems of insufficient sample amount and limited proliferation effect of primary tumor cells in the existing three-dimensional culture scheme, and provides model support for preparing a CTOS biological sample bank and carrying out personalized drug evaluation and screening on cancer patients.
Drawings
FIG. 1 is a CTOS inverted phase contrast micrograph of colorectal cancer tissue; a is CTOS inverted phase contrast micrograph of colorectal cancer tissue cultured in serum-free medium at day 1 in example 7; b is the CTOS inverted phase contrast micrograph of colorectal cancer tissue cultured in the serum-free medium of example 7 at day 7.
FIG. 2 is a CTOS growth curve of colorectal cancer tissue cultured in serum-free medium in example 8.
FIG. 3 is a CTOS inverted phase contrast micrograph of breast cancer tissue; a is the CTOS inverted phase contrast micrograph of the breast cancer tissue cultured in the serum-free medium of example 9 on the 1 st day, and B is the CTOS inverted phase contrast micrograph of the breast cancer tissue cultured in the serum-free medium of example 9 on the 7 th day.
FIG. 4 is a CTOS growth curve of breast cancer tissue cultured in serum-free medium according to example 10.
FIG. 5 is an inverted phase contrast micrograph of gastric cancer tissue CTOS; a is the CTOS inverted phase contrast micrograph of the gastric cancer tissue cultured in the serum-free medium of example 11 on day 1, and B is the CTOS inverted phase contrast micrograph of the gastric cancer tissue cultured in the serum-free medium of example 11 on day 7.
FIG. 6 shows CTOS growth curves of gastric cancer tissues cultured in serum-free medium in example 12.
FIG. 7 is a photograph of a collagen gel drop scan of primary colorectal cancer cultured in serum-free medium at the drug sensitivity detection stage of example 7.
FIG. 8 is a photograph of a collagen gel drop scan of breast cancer tissue CTOS cultured in a serum-free medium at a drug sensitivity test stage in example 9.
FIG. 9 is a photograph of a collagen gel drop scan of the gastric cancer tissue CTOS cultured in the serum-free medium in the drug sensitivity test stage in example 11.
FIG. 10 shows the result of the CTOS drug sensitivity test of colorectal cancer tissue cultured in serum-free medium in example 7.
FIG. 11 shows the result of drug sensitivity test of primary breast cancer cells cultured in serum-free medium in example 9.
FIG. 12 shows the result of the detection of CTOS drug sensitivity of gastric cancer tissues cultured in serum-free medium in example 11.
FIG. 13 is an inverted phase contrast micrograph of primary cancer cells; a is colorectal cancer tissue CTOS obtained by culturing according to the method of example 7 using the serum-free medium of example 1; b is the colorectal cancer tissue CTOS obtained by adopting the serum-free culture medium without adding B27, Heregulin and Activin A in the control example 1 and according to the method in the example 7; c is primary breast cancer cell obtained by culturing according to the method of example 7 using the serum-free medium of example 1; d is primary breast cancer cell obtained by culturing the serum-free culture medium without adding B27, Heregulin and Activin A in the control example 1 according to the method of the embodiment 7; e is gastric cancer tissue CTOS obtained by culturing according to the method of example 7 using the serum-free medium of example 4; f is the gastric cancer tissue CTOS obtained by culturing the control example 2 in serum-free medium without addition of B27, Heregulin, Activin A and Gastrin I according to the method of example 7.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Collagen gel solutionConsists of: fraction 1, 3mg/ml type I collagen solution, fraction 2, 10 XF-12 medium, and fraction 3, NaOH, NaHCO3And mixed liquor consisting of HEPES;
wherein, the component 1: and (2) component: component 3 ═ 8:1:1 (v: v: v);
NaOH、NaHCO3and 50mM NaOH and NaHCO in mixed solution consisting of HEPES3 260mM、HEPES 200mM。
Example 1A serum-free Medium
The components and the final concentration thereof are as follows: basal medium DMEM/F12; BSA, 1.55% (w/v); y27632, 10. mu.M; b27 (50X), 2% (v/v); heregulin, 10 ng/ml; EGF, 50 ng/ml; bFGF, 8 ng/ml; activin A, 10 ng/ml; nicotinamide, 10 mM; NEAA (100X), 1% (v/v); trace Elements A (1000X), 0.1% (v/v); trace Elements B (1000X), 0.1% (v/v); trace Elements C (1000X), 0.1% (v/v); 2-mercaptoethanol, 0.1 mM; primocin, 100. mu.g/ml.
Example 2A serum-free Medium
The components and the final concentration thereof are as follows: basal medium DMEM/F12; BSA, 0.1% (w/v); y27632, 1. mu.M; b27 (50X), 0.1% (v/v); heregulin, 1 ng/ml; EGF, 1 ng/ml; bFGF, 1 ng/ml; activin A, 1 ng/ml; nicotinamide, 1 mM; NEAA (100X), 0.1% (v/v); trace Elements A (1000X), 0.01% (v/v); trace Elements B (1000X), 0.01% (v/v); trace Elements C (1000X), 0.01% (v/v); 2-mercaptoethanol, 0.01 mM; primocin, 50. mu.g/ml.
Example 3A serum-free Medium
The components and the final concentration thereof are as follows: basal medium DMEM/F12; BSA, 10% (w/v); y27632, 50. mu.M; b27 (50X), 10% (v/v); heregulin, 100 ng/ml; EGF, 200 ng/ml; bFGF, 100 ng/ml; activin A, 100 ng/ml; nicotinamide, 100 mM; NEAA (100X), 10% (v/v); trace Elements A (1000X), 1% (v/v); trace Elements B (1000X), 1% (v/v); trace Elements C (1000X), 1% (v/v); 2-mercaptoethanol, 1 mM; primocin, 500. mu.g/ml.
Example 4A serum-free Medium
The components and the final concentration thereof are as follows: basal medium DMEM/F12; BSA, 1.55% (w/v); y27632, 10. mu.M; b27 (50X), 2% (v/v); heregulin, 10 ng/ml; EGF, 50 ng/ml; bFGF, 8 ng/ml; activin A, 10 ng/ml; gastrin I, 10 ng/ml; nicotinamide, 10 mM; NEAA (100X), 1% (v/v); trace Elements A (1000X), 0.1% (v/v); trace Elements B (1000X), 0.1% (v/v); trace Elements C (1000X), 0.1% (v/v); 2-mercaptoethanol, 0.1 mM; primocin, 100. mu.g/ml.
Example 5A serum-free Medium
The components and the final concentration thereof are as follows: basal medium DMEM/F12; BSA, 0.1% (w/v); y27632, 1. mu.M; b27 (50X), 0.1% (v/v); heregulin, 1 ng/ml; EGF, 1 ng/ml; bFGF, 1 ng/ml; activin A, 1 ng/ml; gastrin I, 1 ng/ml; nicotinamide, 1 mM; NEAA (100X), 0.1% (v/v); trace Elements A (1000X), 0.01% (v/v) Trace Elements B (1000X), 0.01% (v/v) Trace Elements C (1000X), 0.01% (v/v); 2-mercaptoethanol, 0.01 mM; primocin, 50. mu.g/ml.
Example 6A serum-free Medium
The components and the final concentration thereof are as follows: basal medium DMEM/F12; BSA, 10% (w/v); y27632, 50. mu.M; b27 (50X), 10% (v/v); heregulin, 100 ng/ml; EGF, 200 ng/ml; bFGF, 100 ng/ml; activin A, 100 ng/ml; gastrin I, 100 ng/ml; nicotinamide, 100 mM; NEAA (100X), 10% (v/v); trace Elements A (1000X), 1% (v/v); trace Elements B (1000X), 1% (v/v); trace Elements C (1000X), 1% (v/v); 2-mercaptoethanol, 1 mM; primocin, 500. mu.g/ml.
Example 7 method of culturing spheroids of tissue origin for colorectal cancer
First, experiment method
1. Culture of colorectal cancer CTOS
(1) The obtained fresh colorectal cancer tumor tissue is transferred to a cell culture dish, and is washed 5 times by using sterile normal saline containing 1% of Antibiotic-Antibiotic, so that non-tumor tissue is removed.
(2) And transferring the treated tumor tissue into a 6cm culture dish, dropwise adding 1mL of DMEM/F-12, and dividing the tumor tissue into small fragments with the diameter of less than 1mm by using a sterile scalpel blade, surgical scissors and surgical forceps.
(3) The minced tumor tissue was transferred to a 50mL centrifuge tube and centrifuged at 1,200rpm for 3 min. Centrifuging, removing supernatant, adding 4.5mL DMEM/F12 and 0.5mL cell dispersing enzyme solution (200 u/mL Collagenase, Type I (Gibco) into the precipitated cells, and digesting for 2h at low speed on a 37 ℃ oscillatorTM17100017)。
(4) The tumor cell clusters which have terminated digestion are filtered through a nylon filter membrane with the aperture of 300 mu m, centrifuged at 1200rpm for 3min, and the supernatant is discarded.
(5) 1ml of the serum-free medium of example 1 was added to the cell pellet and mixed well.
(6) The cell suspension was transferred to a culture flask coated with collagen gel solution and gently shaken to distribute the cell pellet evenly.
(7) Placing the culture flask into an incubator in an inclined state, collecting culture medium at the bottom of the flask, and culturing at 37 deg.C with 5% CO2And (5) culturing. After 3 hours, the flask was supplemented with 4ml of the serum-free medium of example 1, placed horizontally in the incubator again for culture, and the medium was replaced every 2 days. Tumor cell growth was monitored by phase contrast microscopy.
2. Collection of colorectal cancer CTOS
(1) After the culture was completed, the medium was discarded, and 2mL of DMEM/F12 and 100. mu.L of cell dispersing enzyme solution, i.e., Collagenase, Type I (Gibco) having a concentration of 200u/mL, were added to the flask and digested for 15 to 30minTM17100017)。
(2) After termination of digestion, the collected tumor cell clusters were filtered through a cell filter having a pore size of 125 μm and 40 μm in order, and colorectal cancer CTOS retained on the 40 μm cell filter was collected, washed twice with HBSS, and centrifuged at 1,200rpm for 3 min. The supernatant was discarded and the cell pellet was collected for further use.
Second, experimental results
As shown in FIG. 1, FIG. 1A is an inverted phase-contrast micrograph of colorectal cancer tissue CTOS at day 1 in serum-free medium culture, and FIG. 1B is an inverted phase-contrast micrograph of colorectal cancer tissue CTOS at day 7 in serum-free medium culture. The results indicate that colorectal cancer tissue CTOS achieved proliferation in the serum-free medium of example 1.
Example 8 method of culturing spheroids of colorectal cancer tissue origin in culture plates
First, experiment method
1. Culture of colorectal cancer CTOS
(1) The obtained fresh colorectal cancer tumor tissue is transferred to a cell culture dish, and is washed 5 times by using sterile normal saline containing 1% of Antibiotic-Antibiotic, so that non-tumor tissue is removed.
(2) And transferring the treated tumor tissue into a 6cm culture dish, dropwise adding 1mL of DMEM/F-12, and dividing the tumor tissue into small fragments with the diameter of less than 1mm by using a sterile scalpel blade, surgical scissors and surgical forceps.
(3) The minced tumor tissue was transferred to a 50mL centrifuge tube and centrifuged at 1,200rpm for 3 min. Centrifuging, removing supernatant, adding 4.5mL DMEM/F12 and 0.5mL cell dispersing enzyme solution (200 u/mL Collagenase, Type I (Gibco) into the precipitated cells, and digesting for 2h at low speed on a 37 ℃ oscillatorTM17100017)。
(4) The tumor cell clusters which have terminated digestion are filtered through a nylon filter membrane with the aperture of 300 mu m, centrifuged at 1200rpm for 3min, and the supernatant is discarded.
(5) And adding the collagen gel solution into the resuspended cell precipitate, and fully and uniformly mixing to obtain a cell-collagen mixed solution.
(6) Dropping 30 μ L of the cell-collagen mixture onto a 6-well culture plate to obtain collagen gel drops, and incubating in an incubator at 37 deg.C for 1 h.
(7) 3mL of the serum-free medium of example 1 was added, and 5% CO was added at 37 ℃2Culturing in an incubator, and replacing the culture medium every 2 days.
2. Detection of colorectal cancer CTOS
(1) After the culture, the cells were fixed by staining. Staining with neutral red for 2h, washing cells with 4mL PBS for 2 times, each time for 15min, fixing with neutral formalin for 45min, soaking in distilled water for 20min, and air drying.
(2) Scanning analysis is carried out on the collagen gel drops by adopting a culture cell analysis system DR6690 or a Primage image analysis system of Darriy biotechnology, Inc. of Guangzhou, and a growth curve is drawn.
Second, experimental results
As a result, as shown in FIG. 2, the colorectal cancer tissue CTOS was proliferated in the serum-free medium of example 1.
Example 9 method of culturing spheroids of tissue origin for breast cancer
First, experiment method
1. Culture of breast cancer CTOS
(1) The obtained fresh breast cancer tumor tissue is transferred to a cell culture dish, and is washed 5 times by sterile normal saline containing 1% of Antibiotic-Antibiotic, so that non-tumor tissue is removed.
(2) And transferring the treated tumor tissue into a 6cm culture dish, dropwise adding 1mL of DMEM/F-12, and dividing the tumor tissue into small fragments with the diameter of less than 1mm by using a sterile scalpel blade, surgical scissors and surgical forceps.
(3) The minced tumor tissue was transferred to a 50mL centrifuge tube and centrifuged at 1,200rpm for 3 min. Centrifuging, removing supernatant, adding 4.5mL DMEM/F12 and 0.5mL cell dispersing enzyme solution (200 u/mL Collagenase, Type I (Gibco) into the precipitated cells, and digesting at low speed for 1h on a 37 ℃ oscillatorTM17100017)。
(4) The clusters of tumor cells that terminated digestion were filtered through a 300 μm nylon filter, centrifuged at 1200rpm for 3min, and the supernatant was discarded.
(5) 1ml of the serum-free medium of example 1 was added to the cell pellet and mixed well.
(6) The cell suspension was transferred to a culture flask coated with collagen gel solution and gently shaken to distribute the cell pellet evenly.
(7) Placing the culture flask into an incubator in an inclined state, collecting culture medium at the bottom of the flask, and culturing at 37 deg.C with 5% CO2And (5) culturing. After 3 hours, the flask was supplemented with 4ml of the serum-free medium of example 1, placed horizontally in the incubator again for culture, and the medium was replaced every 2 days. Tumor cell growth was monitored by phase contrast microscopy.
2. Collection of breast cancer CTOS
(1) After the culture is finished, the culture medium is discarded, 2mL of DMEM/F12 and 100 mu L of cell dispersing enzyme solution are added into the culture flask for digestion for 15-30 min, wherein the cell dispersing enzyme solution is Collagenase, Type I (Gibco) with the concentration of 200u/mLTM17100017)。
(2) After the digestion was terminated, the collected tumor cell clusters were filtered through a cell filter having a pore size of 125 μm and 40 μm in this order, and the breast cancer CTOS retained on the 40 μm cell filter was collected, washed twice with HBSS, and centrifuged at 1,200rpm for 3 min. The supernatant was discarded and the cell pellet was collected for further use.
Second, experimental results
As shown in FIG. 3, FIG. 3A is an inverted phase-contrast micrograph of breast cancer tissue CTOS cultured in a serum-free medium on day 1, and FIG. 3B is an inverted phase-contrast micrograph of breast cancer tissue CTOS cultured in a serum-free medium flask on day 7. The results show that breast cancer tissue CTOS proliferated in the serum-free medium of example 1.
Example 10 method of culturing spheroids of tissue origin for breast cancer
First, experiment method
1. Culture of breast cancer CTOS
(1) The obtained fresh breast cancer tumor tissue is transferred to a cell culture dish, and is washed 5 times by sterile normal saline containing 1% of Antibiotic-Antibiotic, so that non-tumor tissue is removed.
(2) And transferring the treated tumor tissue into a 6cm culture dish, dropwise adding 1mL of DMEM/F-12, and dividing the tumor tissue into small fragments with the diameter of less than 1mm by using a sterile scalpel blade, surgical scissors and surgical forceps.
(3) The minced tumor tissue was transferred to a 50mL centrifuge tube and centrifuged at 1,200rpm for 3 min. Centrifuging, removing supernatant, adding 4.5mL DMEM/F12 and 0.5mL cell dispersing enzyme solution (200 u/mL Collagenase, Type I (Gibco) into the precipitated cells, and digesting for 2h at low speed on a 37 ℃ oscillatorTM17100017)。
(4) The tumor cell clusters which have terminated digestion are filtered through a nylon filter membrane with the aperture of 300 mu m, centrifuged at 1200rpm for 3min, and the supernatant is discarded.
(5) And adding the collagen gel solution into the resuspended cell precipitate, and fully and uniformly mixing to obtain a cell-collagen mixed solution.
(6) Dropping 30 μ L of the cell-collagen mixture onto a 6-well culture plate to obtain collagen gel drops, and incubating in an incubator at 37 deg.C for 1 h.
(7) 3mL of the serum-free medium of example 1 was added, and 5% CO was added at 37 ℃2Culturing in an incubator, and replacing the culture medium every 2 days.
2. Detection of breast cancer CTOS
(1) After the culture, the cells were fixed by staining. Staining with neutral red for 2h, washing cells with 4mL PBS for 2 times, each time for 15min, fixing with neutral formalin for 45min, soaking in distilled water for 20min, and air drying.
(2) Scanning analysis is carried out on the collagen gel drops by adopting a culture cell analysis system DR6690 or a Primage image analysis system of Darriy biotechnology, Inc. of Guangzhou, and a growth curve is drawn.
Second, experimental results
The results are shown in FIG. 4. The results show that breast cancer tissue CTOS proliferated in the serum-free medium of example 1.
Example 11 method for culturing spheroids derived from gastric cancer tissue in culture flask
First, experiment method
1. Culture of gastric cancer CTOS
(1) The obtained fresh gastric cancer tumor tissue is transferred to a cell culture dish, and is washed 5 times by sterile normal saline containing 1% of antimicrobial-antimicrobial, so that non-tumor tissue is removed.
(2) And transferring the treated tumor tissue into a 6cm culture dish, dropwise adding 1mL of DMEM/F-12, and dividing the tumor tissue into small fragments with the diameter of less than 1mm by using a sterile scalpel blade, surgical scissors and surgical forceps.
(3) The minced tumor tissue was transferred to a 50mL centrifuge tube and centrifuged at 1,200rpm for 3 min. Centrifuging, removing supernatant, adding 4.5mL DMEM/F12 and 0.5mL cell dispersing enzyme solution into the precipitated cells, placing on a 37 ℃ oscillator, digesting for 2h with low speed oscillation,the cell dispersing enzyme solution is Collagenase, Type I (Gibco) with the concentration of 200u/mlTM17100017)。
(4) The tumor cell clusters which have terminated digestion are filtered through a nylon filter membrane with the aperture of 300 mu m, centrifuged at 1200rpm for 3min, and the supernatant is discarded.
(5) 1ml of the serum-free medium of example 4 was added to the cell pellet and mixed well.
(6) The cell suspension was transferred to a culture flask coated with collagen gel solution and gently shaken to distribute the cell pellet evenly.
(7) Placing the culture flask into an incubator in an inclined state, collecting culture medium at the bottom of the flask, and culturing at 37 deg.C with 5% CO2And (5) culturing. After 3 hours, the flask was supplemented with 4ml of the serum-free medium of example 4, placed horizontally in the incubator again for culture, and the medium was replaced every 2 days. Tumor cell growth was monitored by phase contrast microscopy.
2. Collection of gastric carcinoma CTOS
(1) After the culture is finished, the culture medium is discarded, 2mL of DMEM/F12 and 100 mu L of cell dispersing enzyme solution are added into the culture flask for digestion for 15-30 min, wherein the cell dispersing enzyme solution is Collagenase, Type I (Gibco) with the concentration of 200u/mLTM17100017)。
(2) After the digestion was terminated, the collected tumor cell clusters were filtered through a cell filter having a pore size of 125 μm and 40 μm in this order, and gastric cancer CTOS retained on the 40 μm cell filter was collected, washed twice with HBSS, and centrifuged at 1,200rpm for 3 min. The supernatant was discarded and the cell pellet was collected for further use.
Second, experimental results
As shown in fig. 5, fig. 5A is an inverted phase contrast micrograph of gastric cancer tissue CTOS on day 1 in serum-free medium culture, and fig. 5B is an inverted phase contrast micrograph of gastric cancer cells on day 7 in serum-free medium culture. The results showed that the gastric cancer tissue CTOS proliferated in the serum-free medium of example 4.
Example 12 method for culturing spheroids derived from gastric cancer tissue in culture plate
First, experiment method
1. Culture of gastric cancer CTOS
(1) The obtained fresh gastric cancer tumor tissue is transferred to a cell culture dish, and is washed 5 times by sterile normal saline containing 1% of antimicrobial-antimicrobial, so that non-tumor tissue is removed.
(2) And transferring the treated tumor tissue into a 6cm culture dish, dropwise adding 1mL of DMEM/F-12, and dividing the tumor tissue into small fragments with the diameter of less than 1mm by using a sterile scalpel blade, surgical scissors and surgical forceps.
(3) The minced tumor tissue was transferred to a 50mL centrifuge tube and centrifuged at 1,200rpm for 3 min. Centrifuging, removing supernatant, adding 4.5mL DMEM/F12 and 0.5mL cell dispersing enzyme solution (200 u/mL Collagenase, Type I (Gibco) into the precipitated cells, and digesting for 2h at low speed on a 37 ℃ oscillatorTM17100017)。
(4) The tumor cell clusters which have terminated digestion are filtered through a nylon filter membrane with the aperture of 300 mu m, centrifuged at 1200rpm for 3min, and the supernatant is discarded.
(5) And adding the collagen gel solution into the resuspended cell precipitate, and fully and uniformly mixing to obtain a cell-collagen mixed solution.
(6) Dropping 30 μ L of the cell-collagen mixture onto a 6-well culture plate to obtain collagen gel drops, and incubating in an incubator at 37 deg.C for 1 h.
(7) 3mL of the serum-free medium of example 4 was added, and 5% CO was added at 37 ℃2Culturing in an incubator, and replacing the culture medium every 2 days.
2. Detection of gastric cancer CTOS
(1) After the culture, the cells were fixed by staining. Staining with neutral red for 2h, washing cells with 4mL PBS for 2 times, each time for 15min, fixing with neutral formalin for 45min, soaking in distilled water for 20min, and air drying.
(2) Scanning analysis is carried out on the collagen gel drops by adopting a culture cell analysis system DR6690 or a Primage image analysis system of Darriy biotechnology, Inc. of Guangzhou, and a growth curve is drawn.
Second, Experimental methods
As a result, as shown in fig. 6, the gastric cancer tissue CTOS proliferated in the serum-free medium of example 4.
Example 13 drug sensitivity assay for CTOS
First, experiment method
The CTOS cultured in vitro in examples 7, 9 and 11 was subjected to drug sensitivity detection using a collagen gel drop embedding culture drug sensitivity detection method.
(1) Adding collagen gel solution to adjusted cell density (2 × 10)5cells/mL), and mixing well to obtain a cell-collagen mixture. Wherein, the collagen gel mixed solution comprises: a, I type collagen solution; b, 10 times concentration of F-12 culture medium; c, NaOH, NaHCO3And a reconstitution buffer comprising HEPES; a: B: C ═ 8:1:1 (v: v: v).
(2) 30 μ L of the cell-collagen mixture was dropped onto a 6-well plate to prepare a collagen gel drop, and the gel drop was incubated in an incubator at 37 ℃ for 1 hour.
(3) 3mL DF10 medium was added at 37 ℃ with 5% CO2Culturing in an incubator for 24 h.
(4) After the culture was completed, the drug group was dosed, fixed by staining at 0-time, and the negative control (control) group without drug was not treated.
(5) After the drug exposure was complete, the drug-containing medium was removed, 4mL of DMEM/F-12 medium was added to each well, and the wells were washed at 37 ℃ for 15min with low-speed shaking. The washing was repeated once.
(6) After removal of the anti-cancer drug, 3mL serum-free medium was added to each well at 37 ℃ with 5% CO2Culturing in an incubator. The medium was changed every 3 days for a total of 7 days.
(7) Staining was fixed on day 8. Staining with neutral red for 2h, washing cells with 4mL PBS for 2 times, each time for 15min, fixing with neutral formalin for 45min, soaking in distilled water for 20min, and air drying.
(8) Scanning analysis of the collagen gel drops was performed using the cultured cell analysis system DR6690 or Primage image analysis system of dary biotechnology, guangzhou.
Second, experimental results
The results are shown in fig. 7 to 12, and fig. 7 is a collagen gel drop scanning picture of the colorectal cancer tissue CTOS cultured in the serum-free medium in the drug sensitivity detection stage in example 7; FIG. 8 is a photograph of a collagen gel drop scan of breast cancer tissue CTOS cultured in a serum-free medium at a drug sensitivity detection stage in example 8; FIG. 9 is a photograph of the gel drop scan of the collagen of the gastric cancer tissue CTOS cultured in the serum-free medium in the drug sensitivity test stage in example 9; FIG. 10 shows the result of detecting CTOS drug sensitivity of colorectal cancer tissue cultured in serum-free medium in example 7; FIG. 11 shows the result of CTOS drug sensitivity test of breast cancer tissues cultured in serum-free medium in example 8; FIG. 12 shows the result of the detection of CTOS drug sensitivity of gastric cancer tissue cultured in serum-free medium in example 9. The result shows that the method for detecting the drug sensitivity of the collagen gel drop embedding culture is suitable for detecting the drug sensitivity of CTOS cultured in vitro, and the activity of the CTOS of colorectal cancer tissues, the CTOS of breast cancer tissues and the CTOS of gastric cancer tissues is gradually reduced along with the increase of the contact concentration of L-OHP, GEM and CDDP drugs.
Comparative example 1A serum-free Medium
The components and the final concentration thereof are as follows: basal medium DMEM/F12; BSA, 1.55% (w/v); y27632, 10. mu.M; EGF, 50 ng/ml; bFGF, 8 ng/ml; nicotinamide, 10 mM; NEAA (100X), 1% (v/v); trace Elements A (1000X), 0.1% (v/v); trace Elements B (1000X), 0.1% (v/v); trace Elements C (1000X), 0.1% (v/v); 2-mercaptoethanol, 0.1 mM; primocin, 100. mu.g/ml.
Comparative example 2A serum-free Medium
The components and the final concentration thereof are as follows: basal medium DMEM/F12; BSA, 1.55% (w/v); y27632, 10. mu.M; EGF, 50 ng/ml; bFGF, 8 ng/ml; nicotinamide, 10 mM; NEAA (100X), 1% (v/v); trace Elements A (1000X), 0.1% (v/v); trace Elements B (1000X), 0.1% (v/v); trace Elements C (1000X), 0.1% (v/v); 2-mercaptoethanol, 0.1 mM; primocin, 100. mu.g/ml.
Comparative example 3
First, experiment method
Colorectal cancer tissue CTOS and breast cancer tissue CTOS were obtained by culturing according to the methods of example 7 and example 9, respectively, using the serum-free medium of comparative example 1, and examined to obtain inverted phase contrast micrographs. Colorectal cancer tissue CTOS and breast cancer tissue CTOS obtained by culturing according to example 7 and example 9 using the serum-free medium of example 1 were used as controls.
Second, experimental results
Experimental results as shown in fig. 13, fig. 13A is the colorectal cancer tissue CTOS obtained by culturing according to the method of example 7 using the serum-free medium of example 1, and fig. 13B is the colorectal cancer tissue CTOS obtained by culturing according to the method of example 7 using the serum-free medium of comparative example 1 without adding B27, Heregulin, and Activin a, and the results show that the serum-free medium of example 1 is more advantageous for the proliferation of the colorectal cancer tissue CTOS.
Experimental results as shown in fig. 13, fig. 13C is primary desmotic breast cancer cells obtained by culturing according to the method of example 7 using the serum-free medium of example 1, and fig. 13D is primary desmotic breast cancer cells obtained by culturing according to the method of example 7 using the serum-free medium of comparative example 1 without adding B27, Heregulin, and Activin a, and the results show that the serum-free medium of example 1 is more advantageous for the clustering and proliferation of breast cancer tissue CTOS.
Comparative example 4
First, experiment method
The gastric cancer tissues CTOS were obtained by culturing in the serum-free medium of comparative example 2 according to the method of example 11, and examined to obtain an inverted phase contrast micrograph. The stomach cancer tissue CTOS obtained by culturing according to example 11 using the serum-free medium of example 4 was used as a control.
Second, experimental results
Experimental results as shown in fig. 13, fig. 13E is a colorectal cancer tissue CTOS obtained by culturing according to the method of example 7 using the serum-free medium of example 4, and fig. 13F is a gastric cancer tissue CTOS obtained by culturing according to the method of example 7 using the serum-free medium of comparative example 2 without adding B27, Heregulin, Activin a and Gastrin I, and the results show that the serum-free medium of example 4 is more advantageous for the proliferation of the gastric cancer tissue CTOS.

Claims (10)

1. A serum-free culture medium is characterized by comprising a basal culture medium, serum albumin, a ROCK inhibitor, B27, Heregulin, Activin A, EGF, bFGF, nicotinamide, NEAA, Trace Elements A, Trace Elements B, Trace Elements C and 2-mercaptoethanol.
2. The serum-free medium according to claim 1, wherein the serum albumin is one or a mixture of two of bovine serum albumin and human serum albumin.
3. The serum-free medium according to claim 1, wherein the serum albumin is Bovine Serum Albumin (BSA), and the concentration of the BSA in the medium is 0.1-10% (w/v).
4. The serum-free medium of claim 1, wherein the ROCK inhibitor is one or more selected from the group consisting of Y27632, thiazovivin, Fasudil, GSK429286A, GSK429286A, RKI-1447, Azaindole 1, GSK269962, Netarsudil, Y-39983, ZINC00881524, Ripasudil hydrochloride dihydrate, and Hydroxyfasuil.
5. The serum-free medium according to claim 1, wherein the ROCK inhibitor is Y27632, and the concentration of Y27632 in the medium is 1-50 μ M.
6. The serum-free culture medium according to claim 1, wherein the basal medium is one of DMEM/F12, Advanced DMEM/F-12, RPMI 1640, DMEM, or F-12.
7. The serum-free medium according to claim 1, wherein the concentration of nicotinamide in the medium is 1-100 mM; the concentration of NEAA is 0.1-10% (v/v); the concentration of Trace Elements A is 0.01-1% (v/v); the concentration of Trace Elements B is 0.01-1% (v/v); the concentration of Trace Elements C is 0.01-1% (v/v); the concentration of the 2-mercaptoethanol is 0.01-1 mM; the concentration of B27 in the culture medium is 0.1-10% (v/v); the concentration of Heregulin is 1-100 ng/ml; the concentration of Activin A is 1-100 ng/ml; the concentration of the EGF is 1-200 ng/ml; the concentration of bFGF is 1-100 ng/ml.
8. The serum-free medium according to claim 1, wherein the medium further comprises Gastrin I.
9. Use of a serum-free medium according to any one of claims 1 to 8 for the culture of spheroids of cancerous tissue origin.
10. A method for culturing spheroids originated from cancer tissues is characterized in that after digestion treatment of tumor tissues, tissues which are not completely digested are removed, cell suspension is collected, supernatant is discarded by centrifugation, a serum-free culture medium according to any one of claims 1 to 8 is added into cell precipitates, the mixture is uniformly mixed and transferred into a culture container coated with collagen gel solution, or the mixture is uniformly mixed with the collagen gel solution and dripped into the culture container for culture, and after the culture is finished, the culture medium is discarded for digestion.
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WO2023123299A1 (en) * 2021-12-31 2023-07-06 Beijing Theraxyte Bioscience Co., Ltd. Compositions and methods for culturing stem cells

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