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CN112608899B - Application of serum-free culture medium in culturing spheroids of cancer tissue origin - Google Patents

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

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CN112608899B
CN112608899B CN202011324214.6A CN202011324214A CN112608899B CN 112608899 B CN112608899 B CN 112608899B CN 202011324214 A CN202011324214 A CN 202011324214A CN 112608899 B CN112608899 B CN 112608899B
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cancer tissue
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trace elements
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CN112608899A (en
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万勇
徐中华
何志华
李明
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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 for the spheroid of the cancer tissue origin, optimize the composition and the preparation method of the culture medium in the original culture method, provide a stable and controllable serum-free culture scheme, solve the problems of insufficient sample size and limited proliferation effect of the primary tumor cells in the existing three-dimensional culture scheme, and provide model support for preparing a CTOS biological sample library and carrying out personalized medicine evaluation and screening for cancer patients.

Description

Application of serum-free culture medium in culturing spheroids of cancer tissue origin
Technical Field
The invention relates to the technical field of cell culture, in particular to application of a serum-free culture medium in culturing spheroids of cancer tissue origin.
Background
Malignant tumor is a common disease threatening human health, and occupies the first three main causes of death in human disease, accounting for one fourth of the causes of death in all diseases. Along with the development of economy and the improvement of living standard, the incidence of tumors is in an annual rising situation, and the incidence and death number of tumors worldwide are expected to reach 2640 ten thousand and 1700 ten thousand respectively in 2030. The treatment of tumor mainly depends on surgical operation, radiotherapy and chemotherapy, targeted treatment, immunotherapy and other technical means. Under the great background of the global great development of accurate medical technology, the development of new anticancer drugs is increasingly important, and more new anticancer drugs are going from laboratory research to clinical application. On one hand, we search for drug targets by 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 commercial 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. Especially in recent years, the rapid development of the three-dimensional cell culture technology is hopeful to be applied to preclinical research of medicines, and can provide basis for clinicians to formulate personalized diagnosis and treatment schemes.
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 structures. Animal models are capable of assessing the efficacy of candidate drugs in a relatively human microenvironment consisting of matrix and host cells (including fibroblasts, endothelial cells, blood cells and immune cells), which models enable the evaluation of pharmacodynamics and pharmacokinetics. The human tumor xenograft (PDX) model refers to a xenograft model in which fresh tumor tissue or tumor cells of a patient are transplanted into an immunodeficient mouse by in situ or ex situ methods, and the like, and grow in dependence 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 can be maintained 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 have shown that the PDX model can be used as a surrogate model for specific patients for screening anticancer drugs to guide personalized treatment of tumors. Compared with a two-dimensional culture cell line, the tumor cells in P DX can retain the original tumor cell characteristics and are closer to the reality of in-vivo cells. However, animal models involve problems such as individual differences of animals, immune rejection and the like, and have the defects of low and unstable neoplasia rate, long modeling time, high price, difficulty in establishing a high-flux platform and the like, and a great deal of scientific research is still needed to solve the problems.
The Cancer Tissue Origin Spheroid (CTOS) is a cell sphere composed of primary tumor cells, is used as a three-dimensional culture model of the primary tumor cells, has the advantages of being close to the cell biological characteristics of in-vivo primary tissues, high in culture success rate, storable and the like, and is an ideal in-vitro drug detection model. The Kondo J group 2011 first discloses a CTOS preparation method (Kondo, J umei, et al proceedings of the National Academy of Sciences,2011, 108.15:6235-6240.) that does not require the tumor cells to be dispersed into individual cells, and maintains contact with each other between the tumor cells throughout the enzymatic digestion and cell culture of cell-matrix interactions. Under milder conditions, the tumor tissue is incompletely digested into cell clusters, and the generated cell clusters and cells pass through a mesh filter, and the cell clusters captured by the filter are further cultured. Further culture in embryonic stem cell medium, these tissue digested primary cells can rapidly form spheroids within 24 hours. Because anoikis is inhibited, the cell death quantity 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 methods still have certain disadvantages, such as large cell loss during the preparation of CTOS; the selection of CTOS mainly depends on manual operation, and is time-consuming, labor-consuming and low in flux; CTOS culture proliferation effect is limited, proliferation is slow, and limited samples are collected for tumor tissue quantity, so that clinical application of the CTOS culture proliferation effect is still limited.
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 spheroids of cancer tissue origin. The invention improves the original method, optimizes the formula of the culture medium, and is more beneficial to the proliferation of tumor cells; the single cell filtration and removal is adjusted to CTOS culture, so that the cell spheroids can be obtained as much as possible; when CTOS is cultured, the CTOS is paved in a culture container coated with a collagen gel solution or directly uniformly mixed in the collagen gel solution, the CTOS is not required to be selected one by one, and the operation is fast and convenient.
It is a first object of the present invention to provide a serum-free medium.
A second object of the present invention is to provide the use of any one of the serum-free media in the culture of spheroids of cancerous tissue origin.
A third object of the present invention is to provide a method for culturing spheroids derived from a cancer tissue.
In order to achieve the above object, the present invention is realized by the following technical scheme:
the invention provides a convenient, rapid and stable culture method for amplifying spheroids of cancer tissue origin, which aims to solve the problems of insufficient sample size and limited proliferation effect of primary tumor cells in the prior art. The culture system of the method adopts a 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 for storage, and can be continuously and stably cultured after resuscitation. 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 the personalized treatment of clinical patients. In addition, various types of tumor models can be obtained by expanding a CTOS biological sample library, and the method can be widely applied to aspects of basic research of tumors, personalized treatment of the tumors, research and development of anti-tumor medicaments and the like, and development of cancer diagnosis and treatment technology is promoted.
Thus, the invention claims a serum-free medium comprising basal medium, serum albumin, ROCK inhibitor, B27, heregulin, activin a, EGF, bFGF, nicotinamide, NEAA, trace Elements a, trace Elements B, trace Elements C, 2-mercaptoethanol.
Trace Elements A is Mediatech 99-182-CI 1000X;
trace Elements B is Mediatech 99-176-CI 1000X;
trace Elements C is Mediteh 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 culture medium is 0.1% -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, netar Sudil (AR-13324), Y-39983, ZINC00881524, ripasudil (K-115) hydrochloride dihydrate and Hydroxyfasudil (HA-1100).
More preferably, the ROCK inhibitor is Y27632, and the concentration of Y27632 in the medium is 1 to 50 μm.
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.
Basal media 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 the Activin A is 1-100 ng/ml; the concentration of 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 10mM.
More preferably, the amount of NEAA in the medium is 1×, i.e. the concentration of NEAA (100×) in the medium is 1% (v/v).
More preferably, the amount of Trace Elements A in the medium is 1×, i.e., the concentration of Trace Elements A (1000×) in the medium is 0.1% (v/v).
More preferably, trace Elements B is used in an amount of 1X in the medium, i.e., the concentration of Trace Elements B (1000X) in the medium is 0.1% (v/v).
More preferably, trace Elements C is used in an amount of 1X in the medium, 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 medium is 0.1mM.
More preferably, the amount of B27 used in the medium is 1X, i.e., the concentration of B27 (50X) in the medium is 2% (v/v).
More preferably, the concentration of Heregulin the medium is 10ng/ml.
More preferably, the concentration of Activin A in the medium is 10ng/ml.
More preferably, the concentration of EGF in the medium is 50ng/ml.
More preferably, the concentration of bFGF in the medium is 8ng/ml.
Preferably, the medium further comprises Gastin I.
Preferably, the concentration of Gastin I in the medium is 1-100 nM.
More preferably, the concentration of Gastin I in the medium is 10nM.
The serum-free culture medium is used for primary cells of gastric cancer, and the culture medium is also added with Gastrin I.
Preferably, the medium further comprises an antibiotic.
More preferably, the antibiotic is one or more of Primocin, amphotericin B, ampicillin, penicillin-G, kanamycin, gentamicin, erythromycin, MRA, neomycin, nystatin, polymyxin B, streptomycin sulfate, tetracycline, tylosin, and the like.
More preferably, the antibiotic is Primocin.
Further preferably, the Primocin concentration is 10 to 500. Mu.g/ml.
Even more preferably, the Primocin concentration is 100 μg/ml.
The use of the serum-free medium described above in the culture of spheroids of cancerous tissue origin is also within the scope of the present invention.
Therefore, the invention also claims a method for culturing the spheroid of cancer tissue origin, after the digestion treatment of tumor tissue, removing undigested complete tissue, collecting cell suspension, centrifuging to discard supernatant, adding any one of the serum-free culture mediums into cell sediment, uniformly mixing, transferring to a culture container coated with collagen gel solution, or uniformly mixing the culture container with the collagen gel solution, and dripping the culture container into the culture container for culturing.
The collagen gel solution is prepared from the following components: component 1,3mg/ml type I collagen solution, component 2, 10 XF-12 medium, and component 3, naOH, naHCO 3 A mixed solution composition of HEPES;
wherein, component 1: component 2: component 3 = 8:1:1 (v: v);
NaOH、NaHCO 3 NaOH 50mM and NaHCO in HEPES mixed solution 3 260mM、HEPES 200mM。
Preferably, undigested complete tissue is removed by filtration through a nylon filter with a pore size of 300 μm and the cell suspension is collected.
Preferably, at 37℃5% CO 2 Culturing under the condition.
Preferably, the tumor cells are solid tumor cells of stomach 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 a cancer tissue origin spheroid, optimizes the composition and preparation method of the culture medium in the original culture method, provides a stable and controllable serum-free culture scheme, solves the problems of insufficient sample size 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 library and carrying out personalized medicine evaluation and screening on cancer patients.
Drawings
FIG. 1 is a photograph of a colorectal cancer tissue of CTOS inverted phase contrast microscope; a is a CTOS inverted phase contrast microscope photograph of colorectal cancer tissue cultured on day 1 in serum-free medium of example 7; b is a CTOS inverted phase contrast micrograph of colorectal cancer tissue from day 7 of example 7 serum free medium culture.
FIG. 2 is a CTOS growth curve of colorectal cancer tissue cultured in serum-free medium according to example 8.
FIG. 3 is a photograph of a CTOS inverted phase contrast microscope of breast cancer tissue; a is a CTOS inverted phase contrast micrograph of breast cancer tissue from day 1 of the serum-free medium culture of example 9, and B is a CTOS inverted phase contrast micrograph of breast cancer tissue from day 7 of the serum-free medium culture of example 9.
FIG. 4 is a CTOS growth curve of breast cancer tissue cultured in serum-free medium according to example 10.
FIG. 5 is a photograph of an inverted phase contrast microscope of the stomach cancer tissue CTOS; a is a reversed phase contrast micrograph of gastric cancer tissue CTOS on day 1 of the serum-free medium culture of example 11, and B is a reversed phase contrast micrograph of gastric cancer tissue CTOS on day 7 of the serum-free medium culture of example 11.
FIG. 6 is a graph showing the growth of CTOS in gastric cancer tissue cultured in serum-free medium according to example 12.
FIG. 7 is a drop scan of collagen gel from example 7 at the drug susceptibility testing stage for primary colorectal cancer cultured in serum free medium.
FIG. 8 is a photograph of a drop scan of collagen gel of breast cancer tissue CTOS cultured in serum-free medium in the drug sensitivity test phase of example 9.
FIG. 9 is a photograph of a drop scan of collagen gel of gastric cancer tissue CTOS cultured in serum-free medium in the drug sensitivity test phase of example 11.
FIG. 10 shows the results of CTOS drug sensitivity assays for colorectal cancer tissue cultured in serum-free medium according to example 7.
FIG. 11 shows the results of drug sensitivity assays for primary breast cancer cells cultured in serum-free medium of example 9.
FIG. 12 shows the results of the drug susceptibility test of gastric cancer tissue CTOS cultured in serum-free medium in example 11.
FIG. 13 is an inverted phase contrast microscope photograph of primary cancer cells; a is colorectal cancer tissue CTOS obtained by culturing in the serum-free medium of example 1 according to the method of example 7; b is colorectal cancer tissue CTOS obtained by culturing in serum-free medium of comparative example 1 without adding B27, heregulin, and Activin A according to the method of example 7; c is primary-node breast cancer cells obtained by culturing in the serum-free medium of example 1 according to the method of example 7; d is a primary breast cancer cell obtained by culturing in a serum-free medium without adding B27, heregulin, and Activin A of comparative example 1 according to the method of example 7; e is gastric cancer tissue CTOS obtained by culturing in the serum-free medium of example 4 according to the method of example 7; f is a gastric cancer tissue CTOS obtained by culturing in a serum-free medium of comparative example 2 without adding B27, heregulin, activin A and Gastin I in the same manner as in example 7.
Detailed Description
The invention will be further elaborated in connection with the drawings and the specific embodiments described below, which are intended to illustrate the invention only and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The collagen gel solution is prepared from the following components: component 1,3mg/ml type I collagen solution, component 2, 10 XF-12 medium, and component 3, naOH, naHCO 3 A mixed solution composition of HEPES;
wherein, component 1: component 2: component 3 = 8:1:1 (v: v);
NaOH、NaHCO 3 NaOH 50mM and NaHCO in HEPES mixed solution 3 260mM、HEPES 200mM。
Example 1 serum-free Medium
The components and the final concentration thereof are as follows: basic culture medium DMEM/F12; BSA,1.55% (w/v); y27632, 10. Mu.M; b27 (50×), 2% (v/v); heregulin,10ng/ml; EGF,50ng/ml; bFGF,8ng/ml; activin A,10ng/ml; nicotinamide, 10mM; NEAA (100×), 1% (v/v); trace Elements A (1000×), 0.1% (v/v); trace Elements B (1000×), 0.1% (v/v); trace Elements C (1000×), 0.1% (v/v); 2-mercaptoethanol, 0.1mM; primocin,100 μg/ml.
EXAMPLE 2 serum-free Medium
The components and the final concentration thereof are as follows: basic culture medium DMEM/F12; BSA,0.1% (w/v); y27632, 1. Mu.M; b27 (50X), 0.1% (v/v); heregulin,1ng/ml; EGF,1ng/ml; bFGF,1ng/ml; activin A,1ng/ml; nicotinamide, 1mM; NEAA (100×), 0.1% (v/v); trace Elements A (1000×), 0.01% (v/v); trace Elements B (1000×), 0.01% (v/v); trace Elements C (1000×), 0.01% (v/v); 2-mercaptoethanol, 0.01mM; primocin,50 μg/ml.
EXAMPLE 3 serum-free Medium
The components and the final concentration thereof are as follows: basic culture medium DMEM/F12; BSA,10% (w/v); y27632, 50. Mu.M; b27 (50X), 10% (v/v); heregulin,100ng/ml; EGF,200ng/ml; bFGF,100ng/ml; activin A,100ng/ml; nicotinamide, 100mM; NEAA (100×), 10% (v/v); trace Elements A (1000×), 1% (v/v); trace Elements B (1000×), 1% (v/v); trace Elements C (1000×), 1% (v/v); 2-mercaptoethanol, 1mM; primocin,500 μg/ml.
EXAMPLE 4 serum-free Medium
The components and the final concentration thereof are as follows: basic culture medium DMEM/F12; BSA,1.55% (w/v); y27632, 10. Mu.M; b27 (50×), 2% (v/v); heregulin,10ng/ml; EGF,50ng/ml; bFGF,8ng/ml; activin A,10ng/ml; gastin I,10ng/ml; nicotinamide, 10mM; NEAA (100×), 1% (v/v); trace Elements A (1000×), 0.1% (v/v); trace Elements B (1000×), 0.1% (v/v); trace Elements C (1000×), 0.1% (v/v); 2-mercaptoethanol, 0.1mM; primocin,100 μg/ml.
EXAMPLE 5 serum-free Medium
The components and the final concentration thereof are as follows: basic culture medium DMEM/F12; BSA,0.1% (w/v); y27632, 1. Mu.M; b27 (50X), 0.1% (v/v); heregulin,1ng/ml; EGF,1ng/ml; bFGF,1ng/ml; activin A,1ng/ml; gastin I,1ng/ml; nicotinamide, 1mM; NEAA (100×), 0.1% (v/v); trace Elements A (1000×), 0.01% (v/v) Trace Elements B (1000×), 0.01% (v/v) Trace Elements C (1000×), 0.01% (v/v); 2-mercaptoethanol, 0.01mM; primocin,50 μg/ml.
EXAMPLE 6 serum-free Medium
The components and the final concentration thereof are as follows: basic culture medium DMEM/F12; BSA,10% (w/v); y27632, 50. Mu.M; b27 (50X), 10% (v/v); heregulin,100ng/ml; EGF,200ng/ml; bFGF,100ng/ml; activin A,100ng/ml; gastin I,100ng/ml; nicotinamide, 100mM; NEAA (100×), 10% (v/v); trace Elements A (1000×), 1% (v/v); trace Elements B (1000×), 1% (v/v); trace Elements C (1000×), 1% (v/v); 2-mercaptoethanol, 1mM; primocin,500 μg/ml.
EXAMPLE 7 method of culturing colorectal cancer tissue origin spheroids in culture flask
1. Experimental method
1. Cultivation of colorectal cancer CTOS
(1) The obtained fresh colorectal cancer tumor tissue is transferred into a cell culture dish, and the tumor tissue is removed by washing the tumor tissue with sterile physiological saline containing 1% of antibacterial-antibacterial for 5 times.
(2) The treated tumor tissue was transferred to a 6cm petri dish, 1mL DMEM/F-12 was added dropwise, and the tumor tissue was divided into small pieces smaller than 1mm in diameter with a sterile scalpel, surgical scissors and forceps.
(3) The minced tumor tissue was transferred to a 50mL centrifuge tube and centrifuged at 1,200rpm for 3min. Centrifuging, adding 4.5mL of DMEM/F12 and 0.5mL of cell dispersion enzyme solution, and shaking at low speed on a shaker at 37deg.C for digestion for 2h, wherein the cell dispersion enzyme solution is Collagenase with concentration of 200u/mL, type I (Gibco TM 17100017)。
(4) The digested tumor cell clusters were stopped and filtered through a nylon filter with a pore size of 300 μm, centrifuged at 1200rpm for 3min, and the supernatant was discarded.
(5) To the cell pellet, 1ml of the serum-free medium of example 1 was added and mixed well.
(6) Transferring the cell suspension into a culture flask coated with collagen gel solution, and gently shaking to uniformly distribute the cell mass.
(7) Placing the flask in an incubator with its inclination, and placing culture medium at the bottom of the flask at 37deg.C and 5% CO 2 Culturing. After 3 hours, 4ml of the serum-free medium of example 1 was added to the flask, and the flask was again placed in the incubator to be cultured in a horizontal state, and the medium was replaced every 2 days. The growth of tumor cells was monitored using a phase contrast microscope.
2. Collection of colorectal cancer CTOS
(1) After the completion of the culture, the medium was discarded, and 2mL of DMEM/F12 and 100. Mu.L of a cell-dispersing enzyme solution, which was Collagenase, type I (Gibco) at a concentration of 200u/mL, were added to the flask and digested for 15 to 30 minutes TM 17100017)。
(2) After the termination of digestion, the collected tumor cell clusters were sequentially filtered through a cell filter having a pore size of 125 μm and a cell filter having a pore size of 40 μm, and colorectal cancer CTOS remaining on the cell filter having a pore size of 40 μm was collected, washed twice with HBSS, and centrifuged at 1,200rpm for 3min. The supernatant was discarded and the cell pellet was collected for further use.
2. Experimental results
As a result, FIG. 1A shows a CTOS inverted phase contrast micrograph of colorectal cancer tissue on day 1 in serum-free medium, and FIG. 1B shows a CTOS inverted phase contrast micrograph of colorectal cancer tissue on day 7 in serum-free medium. The results indicate that colorectal cancer tissue CTOS achieved proliferation in serum-free medium of example 1.
EXAMPLE 8 method of culturing colorectal cancer tissue-derived spheroids in culture plates
1. Experimental method
1. Cultivation of colorectal cancer CTOS
(1) The obtained fresh colorectal cancer tumor tissue is transferred into a cell culture dish, and the tumor tissue is removed by washing the tumor tissue with sterile physiological saline containing 1% of antibacterial-antibacterial for 5 times.
(2) The treated tumor tissue was transferred to a 6cm petri dish, 1mL DMEM/F-12 was added dropwise, and the tumor tissue was divided into small pieces smaller than 1mm in diameter with a sterile scalpel, surgical scissors and forceps.
(3) The minced tumor tissue was transferred to a 50mL centrifuge tube and centrifuged at 1,200rpm for 3min. Centrifuging, adding 4.5mL of DMEM/F12 and 0.5mL of cell dispersion enzyme solution, and shaking at low speed on a shaker at 37deg.C for digestion for 2h, wherein the cell dispersion enzyme solution is Collagenase with concentration of 200u/mL, type I (Gibco TM 17100017)。
(4) The digested tumor cell clusters were stopped and filtered through a nylon filter with a pore size of 300 μm, centrifuged at 1200rpm for 3min, and the supernatant was discarded.
(5) Adding the collagen gel solution into the cell sediment after the resuspension, and fully and uniformly mixing to obtain the cell-collagen mixed solution.
(6) The 30. Mu.L of the cell-collagen mixture was dropped onto a 6-well culture plate to prepare a collagen gel drop, which was put into a 37℃incubator to be incubated for 1 hour.
(7) 3mL of the serum-free medium of example 1 was added at 37℃with 5% CO 2 Culturing in incubator, and changing culture medium every 2 days.
2. Detection of colorectal cancer CTOS
(1) After the cultivation is finished, the staining and fixation are carried out. Neutral red staining for 2h, washing cells with 4mL PBS for 2 times, 15min each time, neutral formalin fixation for 45min, soaking in distilled water for 20min, and air drying.
(2) The collagen gel drops were scanned and analyzed using cultured cell analysis system DR6690 or Primage image analysis system from dary biotechnology, inc.
2. Experimental results
As a result, as shown in FIG. 2, colorectal cancer tissue CTOS was proliferated in the serum-free medium of example 1.
Example 9 method of culturing spheroids of breast cancer tissue origin in culture flask
1. Experimental method
1. Culture of breast cancer CTOS
(1) The obtained fresh breast cancer tumor tissue is transferred into a cell culture dish, and the tissue is washed 5 times by sterile physiological saline containing 1% of antibacterial-antibacterial, so as to remove non-tumor tissue.
(2) The treated tumor tissue was transferred to a 6cm petri dish, 1mL DMEM/F-12 was added dropwise, and the tumor tissue was divided into small pieces smaller than 1mm in diameter with a sterile scalpel, surgical scissors and forceps.
(3) The minced tumor tissue was transferred to a 50mL centrifuge tube and centrifuged at 1,200rpm for 3min. Centrifuging, adding 4.5mL of DMEM/F12 and 0.5mL of cell dispersion enzyme solution, and shaking at low speed on a shaker at 37deg.C for digestion for 1 hr, wherein the cell dispersion enzyme solution is Collagenase with concentration of 200u/mL, type I (Gibco TM 17100017)。
(4) The digested tumor cell clusters were stopped and filtered through a 300 μm nylon filter, centrifuged at 1200rpm for 3min, and the supernatant was discarded.
(5) To the cell pellet, 1ml of the serum-free medium of example 1 was added and mixed well.
(6) Transferring the cell suspension into a culture flask coated with collagen gel solution, and gently shaking to uniformly distribute the cell mass.
(7) Placing the flask in an incubator with its inclination, and placing culture medium at the bottom of the flask at 37deg.C and 5% CO 2 Culturing. After 3 hours, 4ml of the serum-free medium of example 1 was added to the flask, and the flask was again placed in the incubator to be cultured in a horizontal state, and the medium was replaced every 2 days. The growth of tumor cells was monitored using a phase contrast microscope.
2. Collection of breast cancer CTOS
(1) After the culture, the culture medium was discarded, and 2mL of DMEM/F12 and 100. Mu.L of a cell-dispersing enzyme solution, which was Collagenase, type I (Gibco TM 17100017)。
(2) After the termination of digestion, the collected tumor cell clusters were sequentially filtered through a cell filter having a pore size of 125 μm and a cell filter having a pore size of 40 μm, and breast cancer CTOS remaining on the cell filter having a pore size of 40 μm was collected, washed twice with HBSS, and centrifuged at 1,200rpm for 3min. The supernatant was discarded and the cell pellet was collected for further use.
2. Experimental results
As a result, FIG. 3 shows the CTOS inverted phase contrast microscope photograph of the breast cancer tissue on day 1 in the serum-free medium, and FIG. 3B shows the CTOS inverted phase contrast microscope photograph of the breast cancer tissue on day 7 in the serum-free medium in the flask. The results indicate that breast cancer tissue CTOS achieved proliferation in serum-free medium of example 1.
EXAMPLE 10 method of culturing spheroids of breast cancer tissue origin in culture plates
1. Experimental method
1. Culture of breast cancer CTOS
(1) The obtained fresh breast cancer tumor tissue is transferred into a cell culture dish, and the tissue is washed 5 times by sterile physiological saline containing 1% of antibacterial-antibacterial, so as to remove non-tumor tissue.
(2) The treated tumor tissue was transferred to a 6cm petri dish, 1mL DMEM/F-12 was added dropwise, and the tumor tissue was divided into small pieces smaller than 1mm in diameter with a sterile scalpel, surgical scissors and forceps.
(3) The minced tumor tissue was transferred to a 50mL centrifuge tube and centrifuged at 1,200rpm for 3min. Centrifuging, adding 4.5mL of DMEM/F12 and 0.5mL of cell dispersion enzyme solution, and shaking at low speed on a shaker at 37deg.C for digestion for 2h, wherein the cell dispersion enzyme solution is Collagenase with concentration of 200u/mL, type I (Gibco TM 17100017)。
(4) The digested tumor cell clusters were stopped and filtered through a nylon filter with a pore size of 300 μm, centrifuged at 1200rpm for 3min, and the supernatant was discarded.
(5) Adding the collagen gel solution into the cell sediment after the resuspension, and fully and uniformly mixing to obtain the cell-collagen mixed solution.
(6) The 30. Mu.L of the cell-collagen mixture was dropped onto a 6-well culture plate to prepare a collagen gel drop, which was put into a 37℃incubator to be incubated for 1 hour.
(7) 3mL of the serum-free medium of example 1 was added at 37℃with 5% CO 2 Culturing in incubator, and changing culture medium every 2 days.
2. Detection of breast cancer CTOS
(1) After the cultivation is finished, the staining and fixation are carried out. Neutral red staining for 2h, washing cells with 4mL PBS for 2 times, 15min each time, neutral formalin fixation for 45min, soaking in distilled water for 20min, and air drying.
(2) The collagen gel drops were scanned and analyzed using cultured cell analysis system DR6690 or Primage image analysis system from dary biotechnology, inc.
2. Experimental results
The results are shown in FIG. 4. The results indicate that breast cancer tissue CTOS achieved proliferation in serum-free medium of example 1.
EXAMPLE 11 method of culturing spheroids derived from stomach cancer tissue in culture flask
1. Experimental method
1. Culture of gastric cancer CTOS
(1) The obtained fresh gastric cancer tumor tissue is transferred into a cell culture dish, and the fresh gastric cancer tumor tissue is washed 5 times by sterile physiological saline containing 1% of antibacterial-antibacterial, so that non-tumor tissue is removed.
(2) The treated tumor tissue was transferred to a 6cm petri dish, 1mL DMEM/F-12 was added dropwise, and the tumor tissue was divided into small pieces smaller than 1mm in diameter with a sterile scalpel, surgical scissors and forceps.
(3) The minced tumor tissue was transferred to a 50mL centrifuge tube and centrifuged at 1,200rpm for 3min. Centrifuging, adding 4.5mL of DMEM/F12 and 0.5mL of cell dispersion enzyme solution, and shaking at low speed on a shaker at 37deg.C for digestion for 2h, wherein the cell dispersion enzyme solution is Collagenase with concentration of 200u/mL, type I (Gibco TM 17100017)。
(4) The digested tumor cell clusters were stopped and filtered through a nylon filter with a pore size of 300 μm, centrifuged at 1200rpm for 3min, and the supernatant was discarded.
(5) To the cell pellet, 1ml of the serum-free medium of example 4 was added and mixed well.
(6) Transferring the cell suspension into a culture flask coated with collagen gel solution, and gently shaking to uniformly distribute the cell mass.
(7) Placing the flask in an incubator with its inclination, and placing culture medium at the bottom of the flask at 37deg.C and 5% CO 2 Culturing. After 3 hours, 4ml of the serum-free medium of example 4 was added to the flask, and the flask was again placed in the incubator to be cultured in a horizontal stateThe medium was changed every 2 days. The growth of tumor cells was monitored using a phase contrast microscope.
2. Collection of gastric cancer CTOS
(1) After the culture, the culture medium was discarded, and 2mL of DMEM/F12 and 100. Mu.L of a cell-dispersing enzyme solution, which was Collagenase, type I (Gibco TM 17100017)。
(2) After the digestion was terminated, the collected tumor cell clusters were sequentially filtered through a cell filter having a pore size of 125 μm and a cell filter having a pore size of 40 μm, and gastric cancer CTOS remaining on the cell filter having a pore size of 40 μm was collected, washed twice with HBSS, and centrifuged at 1,200rpm for 3min. The supernatant was discarded and the cell pellet was collected for further use.
2. Experimental results
As a result, FIG. 5 shows a phase contrast inverted microscope photograph of the gastric cancer tissue CTOS on day 1 in the serum-free medium, and FIG. 5B shows a phase contrast inverted microscope photograph of the gastric cancer cells on day 7 in the serum-free medium. The results indicate that proliferation of gastric cancer tissue CTOS was achieved in serum-free medium of example 4.
EXAMPLE 12 method of culturing spheroids derived from gastric cancer tissue in culture plate
1. Experimental method
1. Culture of gastric cancer CTOS
(1) The obtained fresh gastric cancer tumor tissue is transferred into a cell culture dish, and the fresh gastric cancer tumor tissue is washed 5 times by sterile physiological saline containing 1% of antibacterial-antibacterial, so that non-tumor tissue is removed.
(2) The treated tumor tissue was transferred to a 6cm petri dish, 1mL DMEM/F-12 was added dropwise, and the tumor tissue was divided into small pieces smaller than 1mm in diameter with a sterile scalpel, surgical scissors and forceps.
(3) The minced tumor tissue was transferred to a 50mL centrifuge tube and centrifuged at 1,200rpm for 3min. Centrifuging, adding 4.5mL of DMEM/F12 and 0.5mL of cell dispersion enzyme solution, and shaking at low speed on a shaker at 37deg.C for digestion for 2h, wherein the cell dispersion enzyme solution is Collagenase with concentration of 200u/mL, type I (Gibco TM 17100017)。
(4) The digested tumor cell clusters were stopped and filtered through a nylon filter with a pore size of 300 μm, centrifuged at 1200rpm for 3min, and the supernatant was discarded.
(5) Adding the collagen gel solution into the cell sediment after the resuspension, and fully and uniformly mixing to obtain the cell-collagen mixed solution.
(6) The 30. Mu.L of the cell-collagen mixture was dropped onto a 6-well culture plate to prepare a collagen gel drop, which was put into a 37℃incubator to be incubated for 1 hour.
(7) 3mL of serum-free medium of example 4 was added at 37℃with 5% CO 2 Culturing in incubator, and changing culture medium every 2 days.
2. Detection of gastric cancer CTOS
(1) After the cultivation is finished, the staining and fixation are carried out. Neutral red staining for 2h, washing cells with 4mL PBS for 2 times, 15min each time, neutral formalin fixation for 45min, soaking in distilled water for 20min, and air drying.
(2) The collagen gel drops were scanned and analyzed using cultured cell analysis system DR6690 or Primage image analysis system from dary biotechnology, inc.
2. Experimental method
As a result, as shown in FIG. 6, the proliferation of the gastric cancer tissue CTOS was achieved in the serum-free medium of example 4.
Example 13 drug sensitivity detection of CTOS
1. Experimental method
CTOS cultured in vitro in examples 7, 9 and 11 was subjected to drug susceptibility testing using the collagen gel drop-in culture drug susceptibility testing method.
(1) Adding collagen gel solution to the adjusted cell density (2×10) 5 cells/mL) was thoroughly mixed to obtain a cell-collagen mixture. Wherein, the collagen gel mixed solution comprises: a type A, type I collagen solution; b,10 times concentration F-12 culture medium; c, naOH and NaHCO 3 A reconstitution buffer comprising HEPES; a: B: c=8:1:1 (v: v).
(2) mu.L of the cell-collagen mixture was dropped onto a 6-well culture plate to prepare a collagen gel drop, which was placed in a 37℃incubator to incubate for 1 hour.
(3) 3mL of DF10 medium was added at 37℃with 5% CO 2 Culturing in an incubator for 24 hours.
(4) After the end of the incubation, the drug group was dosed, stained with 0-time and fixed, and the negative control (control) group without drug was not treated.
(5) After the completion of the drug contact, the drug-containing medium was removed, 4mL of DMEM/F-12 medium was added to each well, and the mixture was washed with shaking at 37℃for 15 minutes. The wash was repeated once.
(6) After removal of the anticancer drugs, 3mL of serum-free medium was added to each well, 37℃and 5% CO 2 Culturing in an incubator. The medium was changed every 3 days for a total of 7 days.
(7) Staining was performed on day 8. Neutral red staining for 2h, washing cells with 4mL PBS for 2 times, 15min each time, neutral formalin fixation for 45min, soaking in distilled water for 20min, and air drying.
(8) Collagen gel droplets were scanned using cultured cell analysis system DR6690 or Primage image analysis system, of dary biotechnology, inc.
2. Experimental results
The results are shown in FIGS. 7 to 12, and FIG. 7 is a drop scan of collagen gel of colorectal cancer tissue CTOS cultured in serum-free medium according to example 7 during drug sensitivity detection phase; FIG. 8 is a photograph of a drop scan of collagen gel of breast cancer tissue CTOS cultured in serum-free medium in the drug sensitivity test phase of example 8; FIG. 9 is a photograph of a drop scan of collagen gel of gastric cancer tissue CTOS cultured in serum-free medium in the drug sensitivity test phase of example 9; FIG. 10 is a graph showing the results of CTOS drug sensitivity assays for colorectal cancer tissue cultured using serum-free medium in example 7; FIG. 11 shows the results of CTOS drug sensitivity assays for breast cancer tissue cultured in serum-free medium according to example 8; FIG. 12 shows the results of the drug susceptibility test of gastric cancer tissue CTOS cultured in serum-free medium in example 9. The results show that the collagen gel drip embedding culture drug sensitivity detection method is suitable for the drug sensitivity detection of CTOS cultured in vitro, and the activities of colorectal cancer tissue CTOS, breast cancer tissue CTOS and gastric cancer tissue CTOS gradually decline along with the increase of the drug contact concentration of L-OHP, GEM, CDDP.
Comparative example 1A serum-free Medium
The components and the final concentration thereof are as follows: basic culture medium DMEM/F12; BSA,1.55% (w/v); y27632, 10. Mu.M; EGF,50ng/ml; bFGF,8ng/ml; nicotinamide, 10mM; NEAA (100×), 1% (v/v); trace Elements A (1000×), 0.1% (v/v); trace Elements B (1000×), 0.1% (v/v); trace Elements C (1000×), 0.1% (v/v); 2-mercaptoethanol, 0.1mM; primocin,100 μg/ml.
Comparative example 2A serum-free Medium
The components and the final concentration thereof are as follows: basic culture medium DMEM/F12; BSA,1.55% (w/v); y27632, 10. Mu.M; EGF,50ng/ml; bFGF,8ng/ml; nicotinamide, 10mM; NEAA (100×), 1% (v/v); trace Elements A (1000×), 0.1% (v/v); trace Elements B (1000×), 0.1% (v/v); trace Elements C (1000×), 0.1% (v/v); 2-mercaptoethanol, 0.1mM; primocin,100 μg/ml.
Comparative example 3
1. Experimental method
The serum-free medium of comparative example 1 was used to obtain colorectal cancer tissue CTOS and breast cancer tissue CTOS by culturing in the same manner as in example 7 and example 9, respectively, and detection was performed to obtain inverted phase contrast micrographs. Colorectal cancer tissue CTOS and breast cancer tissue CTOS obtained by culturing in accordance with example 7 and example 9 using the serum-free medium of example 1 were used as controls.
2. Experimental results
The experimental results are shown in FIG. 13, in which FIG. 13A is colorectal cancer tissue CTOS obtained by culturing in the serum-free medium of example 1 according to the method of example 7, and FIG. 13B is colorectal cancer tissue CTOS obtained by culturing in the serum-free medium of comparative example 1 without adding B27, heregulin, and Activin A according to the method of example 7, and the serum-free medium of example 1 is shown to be more advantageous for proliferation of colorectal cancer tissue CTOS.
The experimental results are shown in FIG. 13, in which FIG. 13C is a primary-junction breast cancer cell obtained by culturing in the serum-free medium of example 1 according to the method of example 7, and FIG. 13D is a primary-junction breast cancer cell obtained by culturing in the serum-free medium of comparative example 1 without adding B27, heregulin, and Activin A according to the method of example 7, and the result shows that the serum-free medium of example 1 is more favorable for the agglomeration and proliferation of CTOS in breast cancer tissue.
Comparative example 4
1. Experimental method
The respective cultured cells were subjected to the same procedure as in example 11 using the serum-free medium of comparative example 2 to obtain stomach cancer tissues CTOS, and were examined to obtain inverted phase contrast micrographs. The stomach cancer tissue CTOS obtained by culturing in accordance with example 11 using the serum-free medium of example 4 was used as a control.
2. Experimental results
The experimental results are shown in FIG. 13, in which FIG. 13E is colorectal cancer tissue CTOS obtained by culturing in the serum-free medium of example 4 according to the method of example 7, and FIG. 13F is gastric cancer tissue CTOS obtained by culturing in the serum-free medium of comparative example 2 without adding B27, heregulin, activin A and Gastin I according to the method of example 7, and the result shows that the serum-free medium of example 4 is more advantageous for proliferation of gastric cancer tissue CTOS.

Claims (1)

1. Use of a serum-free medium in the culture of spheroids of cancer tissue origin, characterized in that the cancer tissue is rectal cancer tissue, breast cancer tissue and/or gastric cancer tissue;
culturing rectal cancer tissue and/or breast cancer tissue, said serum-free medium consisting of basal medium DMEM/F12, bovine serum albumin 1.55% w/v, ROCK inhibitor Y27632 10 μ M, B27X 2% v/v, heregulin 10ng/ml, activin A10 ng/ml, EGF 50ng/ml, bFGF 8ng/ml, nicotinamide 10mM, NEAA 100X 1% v/v, trace Elements A1000X 0.1% v/v, trace Elements B1000X 0.1% v/v, trace Elements C1000X 0.1% v/v, 2-mercaptoethanol 0.1mM and Primocin 100 μg/ml;
culturing gastric cancer tissue, wherein the serum-free medium consists of basal medium DMEM/F12, bovine serum albumin 1.55%w/v, ROCK inhibitor Y27632 10 [ mu ] M, B ] 27X 2%v/v, heregulin 10ng/ml, activin A10 ng/ml, EGF 50ng/ml, bFGF 8ng/ml, nicotinamide 10mM, NEAA 100X 1%v/v, trace Elements A1000X 0.1%v/v, trace Elements B1000X 0.1%v/v, trace Elements C1000X 0.1%v/v, 2-mercaptoethanol 0.1mM, primocin 100 [ mu ] g/ml and Gastin I10 ng/ml;
the culture method of the cancer tissue origin spheroid comprises the steps of digesting the rectal cancer tissue, the breast cancer tissue and/or the gastric cancer tissue by using DMEM/F12 and cell dispersion enzyme, filtering by using a nylon filter membrane with the aperture of 300 mu m, removing undigested complete tissue, collecting cell suspension, centrifuging, discarding supernatant, adding a serum-free culture medium into cell precipitation, uniformly mixing, transferring into a culture container coated with a collagen gel solution, or uniformly mixing the culture container with the collagen gel solution, dropwise adding the culture container into the culture container for culture, and discarding the culture medium for digestion after the culture is finished.
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