CN109207428B - Method for separating and culturing circulating tumor cells - Google Patents
Method for separating and culturing circulating tumor cells Download PDFInfo
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Abstract
The invention provides a method for separating and culturing circulating tumor cells, a method for screening drugs, a method for determining the effectiveness of the drugs and a culture medium. The method for separating and culturing the circulating tumor cells comprises the following steps: removing CD45 positive cells in anticoagulated peripheral blood by using a Human CD45Depletion Cocktail kit so as to obtain CD45 negative cell sap; treating the CD45 negative cell sap by using a density gradient centrifugation method, and collecting the cell sap of the upper layer and the middle layer after centrifugation; and culturing the cells in the cell sap. The separation and culture method of the circulating tumor cells can effectively enrich the circulating tumor cells, has high yield and high purity, is simple and convenient to operate, and is suitable for large-scale application.
Description
Technical Field
The present invention relates to the field of biology. In particular, the invention relates to methods of isolating and culturing circulating tumor cells.
Background
Tumor metastasis is the leading cause of tumor treatment failure and death in tumor patients. The malignant tumor cells have metastasis in 4 ways: directly invade to the surrounding tissue, lymphatic transfer, blood circulation transfer and abscission, planting and transferring. The blood circulation metastasis means that tumor cells seep into tissues around a primary tumor and enter blood vessels to form Circulating Tumor Cells (CTCs), and the CTCs are transferred to distant tissues such as lung, liver, brain and the like along with blood flow, and then seep out and adapt to a new microenvironment to form a metastasis. Therefore, early detection of CTC in blood plays an important role in guiding patient prognosis, curative effect evaluation and individualized treatment.
Experiments show that the change conditions of the types and the number of the CTCs of the tumor patients are important basis for researching the progress of tumor diseases, the appearance of the CTCs is closely related to the prognosis of patients with advanced cancer, and the prognosis evaluation of patients with metastatic tumors is the most extensive field of clinical application of CTCs at present. Meanwhile, the detection of CTCs as a simple and less invasive blood test can be obtained at any time and used for evaluating the prognosis of patients.
However, isolation and culture methods for circulating tumor cells are still under investigation.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art.
To this end, in one aspect of the invention, the invention provides a method for isolating and culturing circulating tumor cells. According to an embodiment of the invention, the method comprises: removing CD45 positive cells in anticoagulated peripheral blood by using a Human CD45 deletion Cocktail kit so as to obtain CD45 negative cell sap; treating the CD45 negative cell sap by using a density gradient centrifugation method, and collecting the cell sap of the upper layer and the middle layer after centrifugation; and culturing the cells in the cell sap.
Since CTCs are usually CD45-Thus, the kit is used to remove CD45 from peripheral blood+Cells to avoid extraction interference by non-CTCs. However, the inventors found that CD45 could be removed by using only the kit+Cells, which are still unable to effectively enrich CTCs, are too numerous to be mixed with cells. Then, the inventors performed density gradient centrifugation on the CD45 negative cell sap obtained by the kit treatment, and separated CTCs by utilizing the characteristic that specific gravities of different cell populations in peripheral blood differ. After centrifugation, the liquid was divided into an upper layer, an intermediate layer, and a lower layer, and most of the CTCs were present in the intermediate layer. However, the inventors found that a small amount of CTCs is easily mixed in the supernatant. Therefore, the inventors collected the cell sap of the upper and middle layers, cultured it, and screened circulating tumor cells capable of normal growth. Therefore, the method for separating and culturing the circulating tumor cells can effectively enrich the circulating tumor cells, has high yield and good purity, is simple and convenient to operate, and is suitable for large-scale application.
According to an embodiment of the present invention, the culture medium used for the culture includes: wnt 3a protein, N-acetylcysteine, gastrin, epidermal growth factor, R-spondin1 protein, noggin, fibroblast growth factor 10, TGF- β RI, ALK4 and/or ALK7 inhibitor, antibacterial agent, nicotinamide, DMEM medium and F12 medium.
According to the embodiment of the invention, the final concentration of the Wnt 3a protein is 80-200 ng/mL, the final concentration of N-acetylcysteine is 0.5-2 mM, the final concentration of gastrin is 0.5-2 nM, the final concentration of epidermal growth factor is 30-70 ng/mL, the final concentration of R-spondin1 protein is 300-800 ng/mL, the final concentration of noggin is 80-120 ng/mL, the final concentration of fibroblast growth factor 10 is 5-15 ng/mL, the final concentration of TGF-beta RI, ALK4 and/or ALK7 inhibitor is 1-5 mu M, the final concentration of antibacterial agent is 50-150 mu g/mL, the final concentration of nicotinamide is 5-20 mM, and the volume ratio of DMEM culture medium to F12 culture medium is 1: 1.
According to an embodiment of the invention, the medium further comprises: rock inhibitors.
According to the embodiment of the invention, the final concentration of the Rock inhibitor is 5-10 mu mol/L.
According to an embodiment of the invention, the culturing comprises: mixing cells in the cell sap with liquid Matrigel, adding the obtained mixed solution into a pore plate, and preheating to solidify the Matrigel; adding the culture medium into the solidified matrigel, and culturing for 10-12 days; or mixing the cells in the cell sap with the culture medium, dripping the obtained culture solution into a pore plate with the bottom paved with rat tail-derived collagen I, and culturing for 10-12 days.
According to an embodiment of the invention, the density gradient centrifugation is performed in the following manner: mixing the CD45 negative cell sap with a PBS (phosphate buffer solution) containing 1-5% FBS (fiber-reinforced plastic) to obtain a mixed solution; and (3) paving the mixed solution on the lymphocyte separation solution according to the proportion of (1-5) to 1, and then centrifuging the mixed solution at the rotating speed of 1000-1500 g for 8-15 min at the increasing and decreasing speeds of 1 and 0 respectively.
According to an embodiment of the invention, the peripheral blood is taken from an organism suffering from cancer, including gastric, intestinal, lung, gall bladder or pancreatic cancer.
In yet another aspect of the invention, a method of screening for a drug is provided. According to an embodiment of the invention, the method comprises: contacting the candidate agent with circulating tumor cells; measuring the biological activity of the circulating tumor cells before and after the contacting, wherein a decrease in the biological activity of the circulating tumor cells after the contacting as compared to before the contacting is indicative of the drug candidate as a drug of interest, wherein the circulating tumor cells are isolated and cultured using the aforementioned method for isolating and culturing circulating tumor cells. Therefore, the method according to the embodiment of the invention can accurately screen the drugs for inhibiting the CTCs, thereby achieving the effect of treating the cancer.
According to an embodiment of the invention, the medicament is for the treatment of cancer.
In yet another aspect of the invention, a method of determining the effectiveness of a drug is provided. According to an embodiment of the invention, the method comprises: contacting a drug with the circulating tumor cell, the drug being adapted to reduce the biological activity of the circulating tumor cell; determining the biological activity of the circulating tumor cells before and after the contacting, wherein a decrease in the biological activity of the circulating tumor cells after the contacting as compared to before the contacting is indicative of the effectiveness of the agent, wherein the circulating tumor cells were isolated and cultured using the aforementioned methods for isolating and culturing circulating tumor cells. Therefore, whether the drug has the effect of inhibiting the CTCs can be accurately determined by using the method according to the embodiment of the invention, so that the effect of treating the cancer can be achieved.
According to an embodiment of the invention, the medicament is for the treatment of cancer.
In yet another aspect of the invention, a culture medium is provided. According to an embodiment of the invention, the medium comprises: 80-200 ng/mL Wnt 3a protein; 0.5-2 mM N-acetylcysteine; 0.5-2 nM of gastrin; 30-70 ng/mL of epidermal growth factor; 300-800 ng/mL of R-spondin1 protein; 80-120 ng/mL noggin; 5-15 ng/mL fibroblast growth factor 10; 1-5 μ M of a TGF- β RI, ALK4 and/or ALK7 inhibitor; 50-150 mug/mL of an antibacterial agent; 5-20 mM nicotinamide; and DMEM medium and F12 medium at a volume ratio of 1: 1. Thus, circulating tumor cells can be normally grown using the culture medium according to an embodiment of the present invention.
According to an embodiment of the invention, the medium further comprises: rock inhibitors.
According to the embodiment of the invention, the concentration of the Rock inhibitor is 5-10 mu mol/L.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a schematic flow diagram of a method for isolating and culturing circulating tumor cells according to one embodiment of the present invention;
FIG. 2 shows a schematic flow diagram of a method of screening for a drug according to one embodiment of the present invention;
FIG. 3 shows a schematic flow diagram of a method of determining the effectiveness of a drug according to one embodiment of the present invention;
FIG. 4 shows a 4 × micrograph of matrigel-cultured circulating tumor cells according to one embodiment of the invention;
FIG. 5 shows a 4 × micrograph of matrigel-cultured circulating tumor cells of a positive control group according to one embodiment of the present invention;
FIG. 6 shows a 4 × micrograph of matrigel-cultured circulating tumor cells of a negative control group according to one embodiment of the present invention;
FIG. 7 shows a 4 × micrograph of collagen-coated basal plate cultured circulating tumor cells according to one embodiment of the invention;
FIG. 8 shows a schematic diagram of an analysis of the effect of a ROCK inhibitor on the growth of circulating tumor cells, according to one embodiment of the present invention;
FIG. 9 shows a schematic of a drug sensitivity assay according to one embodiment of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
The present invention proposes a method for separating and culturing circulating tumor cells, a method for screening drugs, a method for determining the effectiveness of drugs, and a culture medium, which will be described in detail, respectively, below.
Method for separating and culturing circulating tumor cells
In one aspect of the invention, a method for isolating and culturing circulating tumor cells is provided. According to an embodiment of the invention, referring to fig. 1, the method comprises:
removal of CD 45-positive cells with S100 kit
In this step, CD45 positive cells in anticoagulated peripheral blood were removed using a Human CD45 removal Cocktail kit to obtain CD45 negative cell fluid. Since CTCs are usually CD45-Thus, the kit is used to remove CD45 from peripheral blood+Cells to avoid extraction interference by non-CTCs.
According to an embodiment of the invention, the peripheral blood is taken from an organism suffering from cancer, including gastric, intestinal, lung, gall bladder or pancreatic cancer. Therefore, the method of the invention can obtain the circulating tumor cells of the cancer, and has important effects on prognosis judgment, curative effect evaluation and individual treatment.
The species of the living organisms are not strictly limited, and the living organisms can be human, pig, dog, mouse, rabbit, sheep and the like, and can be flexibly selected according to actual conditions.
S200 Density gradient centrifugation
In this step, the CD 45-negative cell sap was treated by density gradient centrifugation, and the cell sap of the upper and middle layers after centrifugation was collected. The inventors found that CD45 could be removed by using the kit alone+Cells, which cannot effectively enrich CTCs, have a large number of mixed cells. Then, the inventors performed density gradient centrifugation on the CD45 negative cell sap obtained by the kit treatment, and separated CTCs by utilizing the characteristic that specific gravities of different cell populations in peripheral blood differ. After centrifugation, the liquid was divided into an upper layer, an intermediate layer, and a lower layer, and most of the CTCs were present in the intermediate layer. However, the inventors found that a small amount of CTCs is easily mixed in the supernatant. Therefore, the inventors collected the cellular fluids of the upper and middle layers in order to improve the yield of CTCs.
According to an embodiment of the invention, the density gradient centrifugation is performed in the following manner: mixing the CD45 negative cell sap with a PBS (phosphate buffer solution) containing 1-5% FBS (FBS) to obtain a mixed solution; and (3) paving the mixed solution on the lymphocyte separation solution according to the proportion of (1-5) to 1, and then centrifuging the mixed solution at the rotating speed of 1000-1500 g for 8-15 min at the increasing and decreasing speeds of 1 and 0 respectively. Thereby, to further isolate the CTCs.
S300 cultivation
In this step, cells in the cell fluid are cultured.
And (3) culturing the collected cells in the cell sap of the upper layer and the middle layer to screen out circulating tumor cells capable of normally growing. Therefore, the method for separating and culturing the circulating tumor cells can effectively enrich the circulating tumor cells, has high yield and good purity, is simple and convenient to operate, and is suitable for large-scale application.
According to an embodiment of the present invention, the culture medium used for the culture includes: wnt 3a protein, N-acetylcysteine (N-acetylcysteine), Gastrin (Gastrin), epidermal cell growth factor (EGF), R-spondin1 protein, Noggin (Noggin), fibroblast growth factor 10(FGF10), TGF- β RI, ALK4 and/or ALK7 inhibitor (A83-01), antibacterial agent (Normocin), Nicotinamide (Nicotinamide), DMEM medium and F12 medium. According to the specific embodiment of the invention, the final concentration of Wnt 3a protein is 80-200 ng/mL, the final concentration of N-acetylcysteine is 0.5-2 mM, the final concentration of gastrin is 0.5-2 nM, the final concentration of epidermal growth factor is 30-70 ng/mL, the final concentration of R-spondin1 protein is 300-800 ng/mL, the final concentration of noggin is 80-120 ng/mL, the final concentration of fibroblast growth factor 10 is 5-15 ng/mL, the final concentration of TGF-beta RI, ALK4 and/or ALK7 inhibitor is 1-5 mu M, the final concentration of antibacterial agent is 50-150 mu g/mL, the final concentration of nicotinamide is 5-20 mM, and the volume ratio of DMEM culture medium to F12 culture medium is 1: 1.
It should be noted that the concentration of each factor in the culture medium described in the present invention is based on the total volume of DMEM medium/F12 medium.
The composition and the proportion of the culture medium are obtained through a large number of experiments by the inventor, the CTCs can normally grow in the culture medium, and other cells have poor growth states in the culture medium, so that the culture medium not only plays the roles of screening and impurity removal so as to purify the CTCs, but also can achieve the purpose of enriching the CTCs.
According to an embodiment of the invention, the medium further comprises: rock inhibitors.
Rho-Rock is a specific signaling pathway that plays a major role in cell colony formation and interaction between stem cells. When this signaling pathway is blocked by a ROCK inhibitor, such as Y-27632, normal colony formation by stem cells is greatly impaired. The inventors surprisingly found that Rock inhibitors can effectively promote the growth of circulating tumor cells to achieve a better enrichment purpose. However, CTCs are poorly grown in media without Rock inhibitors. Therefore, the purpose of optimizing a circulating tumor cell culture system can be further achieved by adding the Rock inhibitor. Wherein, when the final concentration of the Rock inhibitor is 5-10 mu mol/L, the effect is better, and the obtained circulating tumor cells have more quantity and higher purity.
In the present invention, the culture method of CTCs is not strictly limited, and may be flexibly selected according to actual needs, and a 3D culture method is preferably used. For example, mode 1: mixing cells in the cell sap with liquid Matrigel, adding the obtained mixed solution into a pore plate, and preheating to solidify the Matrigel; adding a culture medium into the solidified matrigel, and culturing for 10-12 days; mode 2: and mixing cells in the cell sap with a culture medium, dropwise adding the obtained culture solution into a pore plate with the bottom paved with rat tail-derived collagen I, and culturing for 10-12 days. The inventors found that the culture using Matrigel (mode 1) was more effective than the culture mode using a collagen-coated plate (mode 2).
Method for screening drugs
In another aspect of the invention, a method of screening for a drug is provided. According to an embodiment of the invention, referring to fig. 2, the method comprises:
a100 contacting a candidate drug with circulating tumor cells
In this step, the drug candidate is contacted with the circulating tumor cells.
A200 measurement of biological Activity of circulating tumor cells before and after contact
In this step, the biological activity of the circulating tumor cells before and after the contact is measured, and the reduction in the biological activity of the circulating tumor cells after the contact as compared to before the contact is an indication that the drug candidate is a drug of interest.
According to an embodiment of the present invention, the circulating tumor cells are isolated and cultured using the previous method for isolating and culturing circulating tumor cells.
At present, the development of tumor-targeted drugs mostly takes solid tumors as therapeutic targets, and CTCs are not taken as targeted drugs. The CTCs obtained by the method for separating and culturing the circulating tumor cells can be used for screening drugs with anti-tumor effects. After contacting with CTCs, the medicine with anti-tumor effect can inhibit the growth and metabolism of CTCs, so that the aim of screening anti-tumor medicine can be achieved by detecting the biological activity of CTCs.
In the present invention, the detection index of "biological activity" is not limited strictly, and any measurement can be performed as long as it relates to the metabolic capability of cell growth. For example, the expression level of a gene or protein, the half lethal dose of a cell, and the like.
In addition, the term "screening drugs" used in the present invention should be understood in a broad sense, and may be either the kind of screening drugs, i.e., the drugs suitable for a specific disease; it is also possible to screen the dosage of the drug, which may be different for different patients due to different sensitivities to the same drug.
According to an embodiment of the invention, the medicament is for the treatment of cancer. The CTCs obtained by the method for separating and culturing the circulating tumor cells can be used for screening drugs with anti-tumor effects.
It will be appreciated by those skilled in the art that the features and advantages described above with respect to the method of isolating and culturing circulating tumor cells are equally applicable to the method of screening for drugs and will not be described in detail herein.
Method for determining the effectiveness of a drug
In yet another aspect of the invention, a method of determining the effectiveness of a drug is provided. According to an embodiment of the invention, referring to fig. 3, the method comprises:
p100 contacting the drug with circulating tumor cells
In this step, a drug is contacted with the circulating tumor cells, the drug being adapted to reduce the biological activity of the circulating tumor cells.
P200 measurement of biological Activity of circulating tumor cells before and after contact
In this step, the biological activity of the circulating tumor cells before and after the contact is measured, and the reduction in the biological activity of the circulating tumor cells after the contact as compared with that before the contact is an indication of the effectiveness of the drug, wherein the circulating tumor cells are isolated and cultured by the aforementioned method for isolating and culturing the circulating tumor cells.
A drug with efficacy can decrease the biological activity of circulating tumor cells, thus, contacting the drug with circulating tumor cells can indicate that the drug is effective if the biological activity of the circulating tumor cells decreases after contact.
According to an embodiment of the invention, the medicament is for the treatment of cancer.
It will be appreciated by those skilled in the art that the features and advantages described above in relation to the method of isolating and culturing circulating tumor cells are equally applicable to the method of determining the effectiveness of a drug and will not be described in further detail herein.
Culture medium
In yet another aspect of the invention, a culture medium is provided. According to an embodiment of the invention, the medium comprises: 80-200 ng/mL Wnt 3a protein; 0.5-2 mM N-acetylcysteine; 0.5-2 nM of gastrin; 30-70 ng/mL of epidermal growth factor; 300-800 ng/mL of R-spondin1 protein; 80-120 ng/mL noggin; 5-15 ng/mL fibroblast growth factor 10; 1-5 μ M of a TGF- β RI, ALK4 and/or ALK7 inhibitor; 50-150 mug/mL of an antibacterial agent; 5-20 mM nicotinamide; and DMEM medium and F12 medium at a volume ratio of 1: 1. The CTCs can normally grow in the culture medium, and other cells have poor growth states in the culture medium, so that the culture medium not only achieves the purposes of screening and impurity removal so as to purify the CTCs, but also can achieve the purpose of enriching the CTCs.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
EXAMPLE 1 culturing of peripheral blood circulating tumor cells (3D culture) in tumor patients
1. Enrichment of peripheral blood tumor cells
1) Preparing PBS containing 2% FBS;
2) adding 50 mu l/ml Human CD45Depletion Cocktail into fresh anticoagulated blood taken from a tumor patient, and incubating for 20min at room temperature (15-25 ℃);
3) adding lymphocyte separation liquid (Ficoll) with the same volume as the blood sample into a new centrifuge tube;
4) after incubation at room temperature, adding PBS containing 2% FBS in an equal volume into a blood sample, uniformly mixing, and paving cell sap on lymphocyte separation liquid in a volume ratio of 2: 1;
5)1200g, 10min, the speed of lifting is 1 and 0 respectively, and centrifuging is carried out;
6) after centrifugation, CD 45-negative monocytes in the upper and middle layers were collected and suspended in 3 to 5-fold volume of PBS containing 2% FBS. 300g, 10min, increasing and decreasing speed by 1 and 0, and centrifuging;
7) washing 2 times according to step 6) and counting.
2. Culture of peripheral blood circulation tumor cell of tumor patient
1) The cells obtained by enrichment are equally divided into two parts, wherein one part is added with a proper amount of Matrigel into the enriched cell sediment according to the counting density, and after uniform mixing, the mixture is dripped into a 24-pore plate by 50 mu l/pore. CO 22Preheating for 10min in an incubator until matrigel is solidified;
2) adding culture medium, 37 deg.C, 5% CO2Culturing in an incubator, wherein the components of a culture medium are shown in table 1;
TABLE 1 Medium composition
| Name of reagent | Final concentration |
| Wnt 3a | 100ng/ml |
| N-acetylcysteine | 1mM |
| Gastrin | 1nM |
| EGF | 50ng/ml |
| Rspondin1 | 500ng/ml |
| Noggin | 100ng/ml |
| FGF10 | 10ng/ml |
| A83-01 | 2μM |
| Normocin | 100μg/ml |
| Nicotinamide | 10mM |
| DMEM/F12 | 1:1 |
3) Cell culture medium was changed every 3 days. The growth state of the cells when cultured for 10-12 days is shown in FIG. 4. In this case, circulating tumor cells were extracted from the blood of the patient, and after culturing in matrigel, single tumor cells grew into spherical tumor tissue analogs (PDTA) in the 3D matrix structure, and 7 PDTAs were co-cultured in this case.
EXAMPLE 2 culturing of peripheral blood circulating tumor cells in tumor patients (3D culture)
In this example, circulating tumor cells were enriched and cultured according to the method of example 1, except that the medium is shown in Table 2.
When the cells were cultured for 10 to 12 days, formation of tumor analogs was observed, the growth state of the cells was good, and the formation efficiency and growth of tumor analogs were similar to those in the culture system of example 1, to obtain 6 PDTAs.
TABLE 2 Medium composition
| Name of reagent | Final concentration |
| Wnt 3a | 200ng/ml |
| N-acetylcysteine | 1.2mM |
| Gastrin | 2nM |
| EGF | 60ng/ml |
| Rspondin1 | 800ng/ml |
| Noggin | 120ng/ml |
| FGF10 | 15ng/ml |
| A83-01 | 3μM |
| Normocin | 150μg/ml |
| Nicotinamide | 15mM |
| DMEM/F12 | 1:1 |
Example 3 peripheral blood circulating tumor cell extraction and culture method establishment (3D culture, Positive control)
1. Enrichment of peripheral blood tumor cells
1) Preparing PBS containing 2% FBS;
2) adding 1000 gastric cancer tumor cells into fresh anticoagulated blood taken from normal people, then adding 50 mu l/ml Human CD45 deletion Cocktail into the anticoagulated blood, and incubating for 20min at room temperature (15-25 ℃);
3) adding lymphocyte separation liquid (Ficoll) with the same volume as the blood sample into a new centrifuge tube;
4) after incubation at room temperature, adding PBS containing 2% FBS in an equal volume into a blood sample, uniformly mixing, and paving cell sap on lymphocyte separation liquid in a volume ratio of 2: 1;
5)1200g, 10min, the speed of lifting is 1 and 0 respectively, and centrifuging is carried out;
6) after centrifugation, CD 45-negative monocytes in the upper and middle layers were collected and suspended in 3 to 5-fold volume of PBS containing 2% FBS. 300g, 10min, increasing and decreasing speed by 1 and 0, and centrifuging;
7) washing 2 times according to step 6) and counting.
2. Culture of peripheral blood circulating tumor cells (3D culture)
1) An appropriate amount of Matrigel was added to the enriched cell pellet according to the count density, and after mixing well, the mixture was dropped into a 24-well plate at 50. mu.l/well. CO 22Preheating for 10min in an incubator until matrigel is solidified;
2) adding culture medium, 37 deg.C, 5% CO2Culturing in an incubator. The medium composition is shown in table 1;
3) cell culture medium was changed every 3 days. The growth state of the cells at 10-12 days of culture is shown in FIG. 5, and the efficiency of PDTA formation is about 60%.
Example 4 culture of Normal human peripheral blood circulating tumor cells (3D culture, negative control)
1. Enrichment of peripheral blood tumor cells
1) Preparing PBS containing 2% FBS;
2) adding 50 mu l/ml Human CD45Depletion Cocktail into fresh anticoagulated blood taken from normal people, and incubating for 20min at room temperature (15-25 ℃);
3) adding lymphocyte separation liquid (Ficoll) with the same volume as the blood sample into a new centrifuge tube;
4) after incubation at room temperature, adding PBS containing 2% FBS in an equal volume into a blood sample, uniformly mixing, and paving cell sap on lymphocyte separation liquid in a volume ratio of 2: 1;
5)1200g, 10min, the speed of lifting is 1 and 0 respectively, and centrifuging is carried out;
6) after centrifugation, CD 45-negative monocytes in the upper and middle layers were collected and suspended in 3 to 5-fold volume of PBS containing 2% FBS. 300g, 10min, increasing and decreasing speed by 1 and 0, and centrifuging;
7) washing 2 times according to step 6) and counting.
2. Culture of Normal human peripheral blood circulating tumor cells (3D culture)
1) An appropriate amount of Matrigel was added to the enriched cell pellet according to the count density, and after mixing well, the mixture was dropped into a 24-well plate at 50. mu.l/well. CO 22Preheating for 10min in an incubator until matrigel is solidified;
2) adding culture medium, 37 deg.C, 5% CO2Culturing in an incubator. The composition of the medium is shown in table 1;
3) cell culture medium was changed every 3 days. The growth state of the cells when cultured for 10-12 days is shown in FIG. 6. There were no circulating tumor cells in normal human serum, so no tumor tissue analog (PDTA) formation was seen in the cultures.
EXAMPLE 5 culturing of peripheral blood circulating tumor cells in tumor patients (collagen-coated substrate based culture)
1. Enrichment of peripheral blood circulating tumor cells in neoplastic patients
Circulating tumor cells were enriched and isolated according to the method of example 1.
2. Collagen I coated culture plate, suspension culture of CTCs
1) Laying a layer of collagen I from rat tail at the bottom of a hole of a 24-hole plate, standing at room temperature for about 1 hour, and washing off redundant collagen I by using a DMEM medium;
2) counting half of the cells obtained by enrichment in step 1, suspending the cells to a proper concentration by using a culture medium, and directly dripping the cells on the solidified collagen I at 37 ℃ and 5% CO2Culturing in an incubator, and changing the cell culture medium every 3 days. When cultured for 10 to 12 days, the results are shown in FIG. 7, in which the tumor cells aggregate to form irregularly arranged cell masses, and the tumor cells cultured under such conditions cannot form 3D structures, resulting in variations in the culturing effect.
Example 6 Effect of ROCK inhibitors on growth of circulating tumor cells
After the enriched circulating tumor cells are subjected to passage amplification, two culture media are respectively used for culturing: (1) the organoids obtained from the culture medium used in example 1 are shown in FIGS. 8A, 8B and 8C, (2) the organoids obtained by adding 8. mu. mol/L of ROCK inhibitor (Y27632) to the culture medium are shown in FIGS. 8D, 8E and 8F. In both cases, the initial culture is performed with substantially the same number of organoid species added per cell culture well. After 1 day and 3 days of culture, the organoid numbers under the two culture conditions were counted, respectively, and the results are shown in fig. 8G, indicating that ROCK inhibitor Y27632 can improve the organoid survival rate, thereby promoting the growth of circulating tumor cells.
Example 7 drug sensitivity assay for peripheral blood CTCs
The method for detecting drug sensitivity of CTCs in peripheral blood will be described below by taking CTCs from gastric cancer patients cultured with matrigel in example 1 as an example. The method can be used for detecting the drug sensitivity of the CTCs in the peripheral blood of patients with the gastric cancer, but not limited to the drug sensitivity of the CTCs in the peripheral blood of the patients with the gastric cancer, and can also be used for detecting the drug sensitivity of the CTCs in the peripheral blood of patients with other types of tumors.
1. Passage and plating of peripheral blood CTCs: peripheral blood CTCs cells obtained by the culture of example 1 were digested and replated in a 96-well plate.
2. Drug gradient experiments:
1) the method using gradient dilution: respectively sucking 10 mu L, 5 mu L, 2.5 mu L, 2 mu L and 1 mu L of original concentration or diluted medicine, adding the medicine into a centrifuge tube containing 1mL of CTCs culture medium of a gastric cancer patient, and sucking 0.5mL from the centrifuge tube into a second centrifuge tube filled with 0.5mL of complete culture medium, namely diluting the medicine according to a ratio of 1: 1. Repeating the above method, sequentially diluting to obtain 5 concentrations required by dosing;
2) the control group absorbs 1 mu L of DMSO or DMF, adds into 1mL of CTCs culture medium of gastric cancer patients, each of the drug with one concentration and the experimental control group is provided with 2-3 compound holes, 3 compound holes are additionally provided with CTCs culture medium, DMSO control and DMF control, and the volume of the drug added into each hole is 140 mu L;
3) liquid changing and secondary medicine adding: the first dosing is day 0, the liquid change at day 3 and the second dosing. The dosing concentration is the same as that of the first time;
4) CCK-8 cell activity assay: and on the 5 th day, detecting the OD value of the cells after the drug adding culture by using a CCK8 detection reagent, wherein the OD value reflects the cell activity and the effect of the drug on the analogues, adding the prepared CCK8 detection solution into each hole, uniformly mixing, placing at 37 ℃ for incubation for 3-4 h, and detecting the light absorption values at 450nm and 650nm by using an enzyme labeling instrument. The calculation formula is as follows: ODDelta 450-ODblank650
The rate of influence of drug on organoids (%) (experimental ODDelta-blank ODDelta) x 100%/(control ODDelta-blank ODDelta)
The results are shown in FIG. 9: taking 2 cases of peripheral blood CTCs of gastric cancer patients as examples, the sensitivity of the CTCs to the chemotherapeutic drug oxaliplatin is detected. The results show that the drug sensitivity of different patients to oxaliplatin is different, the half lethal dose IC50 of oxaliplatin to YDY-S-002 patient is 8.9 mu M, and the half lethal dose IC50 of oxaliplatin to YDY-S-004 patient is 31.9 mu M, namely, the sensitivity of YDY-S-002 patient to oxaliplatin is higher than that of YDY-S-004 patient. The results show that the peripheral blood CTCs of different patients have different sensitivities to the same drug, and the CTCs enriched and cultured by the method can be used for detecting the sensitivities of the patients to chemotherapeutic drugs.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (14)
1. A method for isolating and culturing circulating tumor cells, comprising:
removing CD45 positive cells in anticoagulated peripheral blood by using a Human CD45Depletion Cocktail kit so as to obtain CD45 negative cell sap;
treating the CD45 negative cell sap by using a density gradient centrifugation method, and collecting the cell sap of the upper layer and the middle layer after centrifugation; and
culturing cells in the cell fluid;
the culture medium adopted by the culture comprises:
wnt 3a protein, N-acetylcysteine, gastrin, epidermal growth factor, R-spondin1 protein, noggin, fibroblast growth factor 10, TGF- β RI, ALK4 and/or ALK7 inhibitor, antibacterial agent, nicotinamide, DMEM medium and F12 medium.
2. The method according to claim 1, wherein the final concentration of Wnt 3a protein is 80-200 ng/mL, the final concentration of N-acetylcysteine is 0.5-2 mM, the final concentration of gastrin is 0.5-2 nM, the final concentration of epidermal growth factor is 30-70 ng/mL, the final concentration of R-spondin1 protein is 300-800 ng/mL, the final concentration of noggin is 80-120 ng/mL, the final concentration of fibroblast growth factor 10 is 5-15 ng/mL, the final concentration of TGF- β RI, ALK4 and/or ALK7 inhibitor is 1-5 μ M, the final concentration of antibacterial agent is 50-150 μ g/mL, the final concentration of nicotinamide is 5-20 mM, and the volume ratio of DMEM medium to F12 medium is 1: 1.
3. The method of claim 1, wherein the culture medium further comprises: rock inhibitors.
4. The method of claim 3, wherein the Rock inhibitor is present at a final concentration of 5-10 μmol/L.
5. The method of claim 2, wherein the culturing comprises:
mixing cells in the cell sap with liquid Matrigel, adding the obtained mixed solution into a pore plate, and preheating to solidify the Matrigel; adding the culture medium into the solidified matrigel, and culturing for 10-12 days; or
And mixing the cells in the cell sap with the culture medium, dropwise adding the obtained culture solution into a pore plate with the bottom paved with rat tail-derived collagen I, and culturing for 10-12 days.
6. The method of claim 1, wherein the density gradient centrifugation is performed in the following manner:
mixing the CD45 negative cell sap with a PBS (phosphate buffer solution) containing 1-5% FBS (fiber-reinforced plastic) to obtain a mixed solution;
and (3) paving the mixed solution on the lymphocyte separation solution according to the proportion of (1-5) to 1, and then centrifuging the mixed solution at the rotating speed of 1000-1500 g for 8-15 min at the increasing and decreasing speeds of 1 and 0 respectively.
7. The method of claim 1, wherein the peripheral blood is taken from an organism having a cancer, including gastric, intestinal, lung, gallbladder or pancreatic cancer.
8. A method of screening for a drug comprising:
contacting the candidate agent with circulating tumor cells;
measuring the biological activity of the circulating tumor cells before and after said contacting,
a decrease in the biological activity of the circulating tumor cells after the contacting, as compared to before the contacting, is indicative of the drug candidate as a drug of interest,
wherein the circulating tumor cell is isolated and cultured by the method for isolating and culturing a circulating tumor cell according to any one of claims 1 to 7.
9. The method of claim 8, wherein the medicament is for treating cancer.
10. A method of determining the effectiveness of a drug comprising:
contacting a drug with the circulating tumor cell, the drug being adapted to reduce the biological activity of the circulating tumor cell;
measuring the biological activity of the circulating tumor cells before and after said contacting,
a decrease in the biological activity of the circulating tumor cells after the contacting, as compared to before the contacting, is an indication that the drug is effective,
wherein the circulating tumor cell is isolated and cultured by the method for isolating and culturing a circulating tumor cell according to any one of claims 1 to 7.
11. The method of claim 10, wherein the medicament is for treating cancer.
12. Use of a culture medium for culturing circulating tumor cells, wherein the culture medium comprises:
80-200 ng/mL Wnt 3a protein;
0.5-2 mM N-acetylcysteine;
0.5-2 nM of gastrin;
30-70 ng/mL of epidermal growth factor;
300-800 ng/mL of R-spondin1 protein;
80-120 ng/mL noggin;
5-15 ng/mL fibroblast growth factor 10;
1-5 μ M of a TGF- β RI, ALK4 and/or ALK7 inhibitor;
50-150 mug/mL of an antibacterial agent;
5-20 mM nicotinamide; and
DMEM medium and F12 medium at a volume ratio of 1: 1.
13. The use according to claim 12, wherein the culture medium further comprises: rock inhibitors.
14. The use according to claim 13, wherein the Rock inhibitor is present at a concentration of 5-10 μmol/L.
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