CN113373116A - Primary liver cancer cell culture medium, primary liver cancer cell culture method and application thereof - Google Patents

Primary liver cancer cell culture medium, primary liver cancer cell culture method and application thereof Download PDF

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CN113373116A
CN113373116A CN202010160439.6A CN202010160439A CN113373116A CN 113373116 A CN113373116 A CN 113373116A CN 202010160439 A CN202010160439 A CN 202010160439A CN 113373116 A CN113373116 A CN 113373116A
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liver cancer
cells
primary liver
culture medium
primary
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CN113373116B (en
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刘青松
赵明
陈程
王文超
任涛
王黎
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Precedo Pharmaceuticals Co Ltd
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Abstract

The invention provides a primary liver cancer cell culture medium and a primary liver cancer cell culture method for quickly and stably culturing primary liver cancer cells. The primary liver cancer cell culture medium contains glutamine, non-essential amino acid, basic fibroblast growth factor, hepatocyte growth factor, IL-6, epidermal growth factor, insulin, Y27632, N2 additive, B27 additive, Primocin, penicillin and streptomycin, fetal calf serum, optional hydrocortisone and optional R-spondin 1. The cell model obtained by the primary liver cancer cell culture medium and the primary liver cancer cell culture method can be used for evaluating and screening the curative effect of the medicine.

Description

Primary liver cancer cell culture medium, primary liver cancer cell culture method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primary liver cancer cell culture medium and a primary liver cancer cell culture method for in vitro culture or amplification of primary liver cancer cells, and further relates to a method for evaluating and screening curative effects of the cultured cells in medicines and application of the cultured cells.
Background
Primary culture (primary culture), also called primary culture, refers to the first culture of tumor cells or tissues taken directly from the body. The most commonly used primary cultures are tissue block cultures and dispersed cell cultures. The tissue block culture is to transplant the cut tissue blocks directly on the wall of a culture bottle, and culture is carried out after adding a culture medium. In dispersed cell culture, cells are dispersed from a tissue mass by mechanical or chemical means. For example, to isolate the most active free cells from tumor tissue in clinical biopsies or resections, the classical approach is to digest the intercellular conjugates with proteolytic enzymes (e.g., trypsin and collagenase), or to remove the Ca-dependent cell adhesion with metal ion chelators (e.g., EDTA)2+Then, the mixture is mechanically and lightly oscillated to form single cells.
In recent years, supplementary chemotherapy for liver cancer after operation has been paid more attention and accepted by clinicians as a novel supplementary therapy mode, including postoperative TACE therapy, oral drug therapy, etc., but because of lack of standard chemotherapy schemes, conventional empirical chemotherapy neglects individual differences, and has certain blindness, the effect is always poor, and the effective rate of single drug and combined drug is less than 20% (Jendal A, Thadi A, Shailubhai K.hepatocellular Carcinoma: Iology and Current and Future Drugs [ J ]. J Clin Exp Hepatol,2019,9(2): 221-. Although the toxic and side effects of emerging targeted drugs are reduced to a certain extent, the number of the emerging targeted drugs is too small, the treatment cost is high, the effective rate varies with individuals, and the treatment requirements of most patients are difficult to meet. Mainly, because an effective liver cancer drug sensitivity test system is lacked, like a bacterial drug sensitivity test, the in-vitro drug sensitivity result of the liver cancer corresponds to the clinical in-vivo reaction, and accurate chemotherapy cannot be realized, so that the research on the development of liver cancer sensitive drugs is the key treatment.
The High-throughput Drug Sensitivity experiment technology (High-throughput Drug Sensitivity) is a novel Drug Sensitivity detection method, has the advantages of wide Drug coverage rate, High effective rate, strong individuation and the like, and has made an excellent progress in the field of leukemia. Compared with other types of drug sensitivity experimental models (PDO, PDX), the method has the advantages of economy, rapid detection, easy popularization and the like. The bottleneck of the existing technology in the field of liver cancer is mainly the lack of an effective, rapid and stable primary liver cancer cell culture amplification technology.
It has now been found that primary tumor cell lines can be established rapidly and stably using Conditioned Reprogramming Culture (CRC) of trophoblast cells (Xuefeng Liu, EWA Krawczyk, et al. conditional reprogramming and Long-term expansion of normal and tumor cells from human biosciences. Nature Protocols, vol.12, NO.2,2017, 439-451). However, the stable cell line screened in vitro using the conditioned medium (F Meguim) described in this document has a long culture period and cannot meet the requirement of rapid clinical tests. Therefore, a rapid and stable culture medium and a culture method suitable for primary liver cancer cells need to be developed, so that high-throughput drug screening can be performed within two weeks, and clinical rational drug administration can be effectively guided.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a primary liver cancer cell culture medium and a primary liver cancer cell culture method for quickly and stably culturing primary liver cancer cells in vitro. When the primary liver cancer cell culture medium and the primary liver cancer cell culture method are adopted to construct the primary liver cancer cell model, the primary liver cancer cells with the biological characteristics of a liver cancer patient can be obtained, and the method can be applied to new drug screening and in-vitro drug sensitivity detection.
The invention provides a primary liver cancer cell culture medium for culturing primary liver cancer cells. The primary hepatoma cell culture medium of the invention contains a primary medium, glutamine (Glu), non-essential amino acids (NEAA), basic fibroblast growth factor (bFGF), Hepatocyte Growth Factor (HGF), IL-6, epidermal growth factor, insulin, Y27632, N2 additive, B27 additive, Primocin, penicillin and streptomycin, fetal bovine serum, and optionally hydrocortisone, and optionally R-spondin1, wherein the primary medium may be, for example, DMEM/F12, DMEM, F12, or RPMI-1640, preferably DMEM/F12.
Wherein the content of the glutamine is 62.5 ng/mL-1000 ng/mL, preferably 125 ng/mL-1000 ng/mL; the non-essential amino acid is one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the total content of the non-essential amino acid is 0.25-4 μ M, preferably 0.5-4 μ M; the content of the basic fibroblast growth factor is 5ng/ml to 80ng/ml, preferably 20ng/ml to 80 ng/ml; the content of the hepatocyte growth factor is 5ng/ml to 80ng/ml, preferably 20ng/ml to 80 ng/ml; the content of the IL-6 is 1.25ng/ml to 20ng/ml, preferably 5ng/ml to 20 ng/ml; the content of the epidermal growth factor is 2.5 ng/ml-40 ng/ml, preferably 10 ng/ml-40 ng/ml; the content of the insulin is 1.25 ng/ml-20 ng/ml, preferably 2.5 ng/ml-20 ng/ml; the content of the Y27632 is 5-40 mu M, preferably 10-40 mu M; the volume ratio of the N2 additive to the culture medium is 1: 25-1: 400, preferably 1: 25-1: 100; the volume ratio of the B27 additive to the culture medium is 1: 12.5-1: 200, preferably 1: 12.5-1: 50; the content of the Primocin is 40 to 80 mu g/ml; the content of the penicillin is 80U/ml-200U/ml; the streptomycin content is 50-150 mug/ml; the volume ratio of the fetal calf serum to the culture medium is 1: 5-1: 20; the content of the hydrocortisone is 0.4-1.6 mu g/ml; the content of the R-spondin1 is 62.5 ng/mL-1000 ng/mL, preferably 250 ng/mL-1000 ng/mL.
The invention also provides a culture method of the primary liver cancer cells. In the primary liver cancer cell culture method, the primary liver cancer cell is cultured by using the primary liver cancer culture medium.
The invention relates to primary liver cancer cell cultureIn the cultivation method, 5 is multiplied by 104～1.5×105Per cm2The irradiated trophoblast cells are added to the cell density of (a). The irradiated trophoblast cell is a mouse fibroblast, preferably NIH-3T3 or J2-3T 3. The irradiated trophoblast cells are irradiated with gamma rays or X-rays, preferably gamma rays. The irradiation dose is 10-50 Gy.
The primary liver cancer cell can be obtained by the following method: obtained by a liver cancer resection method; obtained by a puncturing method; obtained from ascites of a patient with liver cancer; or from urine of a liver cancer patient.
More specifically, when obtained by a hepatoma resection or puncture procedure, hepatoma tissue samples are collected within half an hour after surgical resection or puncture of a solid tumor patient. The collection method comprises the following steps: under the sterile environment, a liver cancer tissue sample lcm of a non-necrotic part is cut3The cells are placed in 15mL of the primary liver cancer cell culture medium pre-cooled and placed under the common cell culture condition for later use or transported as soon as possible by using an ice bag.
The culture method of the primary liver cancer cells comprises the step of separating and obtaining the primary liver cancer cells from the tissue sample.
Specifically, in the biosafety cabinet, the tissue sample is transferred to a cell culture dish, and washed three times with a medium containing a penicillin/streptomycin solution (abbreviated as a diabody solution) (for example, DMEM/F12 containing a diabody solution, more preferably, with one or more additives selected from the group consisting of glutamine, nonessential amino acids, basic fibroblast growth factor, hepatocyte growth factor, IL-6, hydrocortisone, epidermal growth factor, insulin, Y27632, N2 additive, B27 additive, R-spondin1, and Primocin, and the concentrations of the above additives may be those in the primary liver cancer cell culture medium of the present invention, and hereinafter, the medium is referred to as a cell isolation medium) to wash out blood cells and exfoliated necrotic cells. The treated tissue sample is transferred to a new culture dish, a proper amount of the culture medium for cell separation is dripped, and the tumor tissue is divided into small fragments with the diameter less than lmm by using a sterile scalpel blade, a pair of surgical scissors and a pair of surgical forceps. The minced tumor tissue is transferred to a centrifuge tube for centrifugation, for example, at 1000-1800 rpm for 3-10 minutes. Carefully removing supernatant in the centrifugal tube, uniformly mixing and resuspending the supernatant with tissue digestive juice, and placing the mixture on a constant-temperature shaking table for digestion for at least 3 hours (the digestion time depends on the size of a sample; if the sample is larger than lg, the digestion time is increased to 4-6 hours) by oscillation (the rotating speed can be 230-350 rpm/min); then, centrifuging for 3-10 minutes at 1000-1800 rpm, for example, discarding the supernatant, resuspending an appropriate amount of the culture medium for cell separation of the digested histiocyte, sieving, wherein the cell sieve can be 100 μ M, then centrifuging again, for example, centrifuging for 3-10 minutes at 1000-1800 rpm, discarding the supernatant, observing whether blood cells exist, for example, blood cells exist, adding an appropriate amount of a blood cell lysate, uniformly mixing and cracking, then centrifuging again, for example, centrifuging for 3-10 minutes at 1000-1800 rpm, discarding the supernatant, adding an appropriate amount of the culture medium for primary liver cancer cells of the invention, resuspending and collecting the cells, and using the cells for adherent expansion culture in a culture bottle.
And when the bottle wall is more than 75% full of cells, carrying out conventional subculture or drug sensitive experiment on the cells.
The present invention also provides a method for evaluating the efficacy of a drug for treating liver cancer diseases, which comprises the steps of:
(1) culturing according to the primary liver cancer cell culture method to obtain liver cancer cells;
(2) selecting the drug to be tested at its maximum plasma concentration CmaxFor reference, 2-5 times of CmaxDiluting to different drug concentration gradients for the initial concentration;
(3) digesting the liver cancer cells obtained by the culture in the step (1) into single cell suspension, diluting the single cell suspension by using the primary liver cancer cell culture medium of the invention, and performing dilution according to the ratio of 2 multiplied by 104Per cm2～4×104Per cm2Adding the diluted cell suspension into a multi-hole plate for overnight adherence, and adding the medicine after gradient dilution to adherent cells;
(4) and (5) carrying out cell viability detection.
In the cell viability detection, a cell viability detection reagent is added into each hole, after uniform oscillation, the chemiluminescence intensity value of each hole is measured by a fluorescence microplate reader, a drug quantity-effect curve is drawn according to the measured value, and the inhibition intensity of each drug on the proliferation of the cells is calculated.
The beneficial effects of the invention include:
(1) the success rate of primary liver cancer cell culture is improved and reaches over 90 percent;
(2) ensuring that the in vitro primary cultured liver cancer cells can reproduce the pathological phenotype and heterogeneity of patients from which the primary cells are derived;
(3) the cultured primary liver cancer cells are not interfered by mesenchymal cells such as fibroblasts and fat cells, and purified liver cancer cells can be obtained;
(4) the efficiency of amplifying primary liver cancer cells is high, only 10 cells are needed4The cell number of the grade can be successfully amplified to 10 within about two weeks6Liver cancer cells of magnitude order, the liver cancer cells amplified can also be continuously passed;
(5) the culture cost is controllable: the primary liver cancer culture medium does not need to add expensive factors such as Wnt agonist, BMP inhibitor, FGF10 and the like, and is an improvement on the existing reprogramming culture medium under the condition.
(6) The culture medium and the culture system have the advantages of large number of liver cancer cells obtained by culture, high homogenization degree, and suitability for high-throughput screening of new candidate compounds and high-throughput drug in-vitro sensitivity function test for patients.
By adopting the primary liver cancer cell culture medium of the embodiment, liver cancer cells derived from people including human beings including hepatocellular carcinoma cells, intrahepatic bile duct cancer cells and mixed liver cancer cells can be cultured.
In addition, the cells obtained by the culture method of the embodiment can be applied to basic medical research of primary liver cancer cells, screening of drug response, research and development of new drugs for liver cancer and the like.
Drawings
FIG. 1 is a photograph of primary liver cancer cells isolated from a liver cancer tissue sample under an inverted phase contrast microscope, wherein the primary liver cancer cells are cultured using the primary cell culture medium of the present invention.
FIG. 2 is a comparison of the immunohistochemical staining results of liver cancer cells obtained by culturing primary liver cancer cells isolated from a liver cancer tissue sample using the primary cell culture medium of the present invention and the immunohistochemical staining results of the original tissue section of the tissue sample itself.
FIG. 3 is a graph showing the effect of different concentrations of different factors in primary liver cancer cell culture medium on proliferation of primary liver cancer cells.
FIG. 4 is an inverted phase contrast microscopic photograph of cells obtained by culturing primary liver cancer cells isolated from a liver cancer tissue sample using a conventional conditioned medium (FM) and a primary liver cancer cell culture medium (HCCM) of the present invention, respectively.
FIG. 5 is a graph showing the statistical effect of multiple passages of cells obtained by culturing primary liver cancer cells isolated from a liver cancer tissue sample using a conventional conditioned medium (FM) and a primary liver cancer cell culture medium (HCCM) of the present invention.
FIG. 6 shows the tumorigenesis of liver cancer cells isolated from liver cancer tissue samples in mice cultured using the primary cell culture medium of the present invention.
FIG. 7 shows the dose-effect curves of different chemotherapeutic drugs and target drugs of hepatoma cells obtained by culturing the primary hepatoma cell culture medium of the present invention and the half-maximal inhibition rates obtained by calculation.
Detailed Description
The present invention will be further described with reference to the following description of specific embodiments, which are provided for illustration only, and the scope of the present invention is not limited to these embodiments.
Example 1 acquisition of Primary liver cancer cells and treatment of trophoblast cells
1. Collection of liver cancer tissue samples
Surgical resection or puncture of liver cancer tissue samples in described and consented liver cancer patientsCollecting in the latter half hour. Under the sterile environment, a liver cancer tissue sample lcm of a non-necrotic part is cut3Above, it was placed in a medium containing 15ml of MEM/F12 pre-cooled in a refrigerator at 4 ℃ and labeled.
2. Treatment of liver cancer tissue samples
(1) Taking tissues from a biosafety cabinet, placing the tissues in a 100mm culture dish (Corning, 430167), removing blood-borne tissues, washing 3 times with a culture medium (hereinafter referred to as a double antibody solution, purchased from Corning, Inc., USA) obtained by adding a 1% streptomycin-penicillin solution (hereinafter referred to as a double antibody solution, wherein the streptomycin concentration in the stock solution is 10mg/ml and the penicillin concentration is 10000U/ml) to a DMEM/F12 culture medium (manufacturer: Corning), transferring the solution to a new 100mm culture dish, adding 10ml of a cell separation medium, and mechanically separating the cells with a sterile scalpel, a surgical scissors and tweezers to divide the tissues into small pieces with a diameter of less than lmm;
(2) transferring the minced tissue into a 50mL centrifuge tube, adding 5mL of cell separation culture medium, uniformly mixing, and centrifuging at 1400rpm for 5 minutes;
(3) carefully removing supernatant in a centrifuge tube by using a pipette, adding a cell separation culture medium and 2X digestive enzyme (containing collagenase II (1mg/mL) (Sigma of the manufacturer) and collagenase IV (1mg/mL) (Sigma of the manufacturer)) prepared according to the volume ratio of 1:1, uniformly mixing and re-suspending, adding 10mL digestive enzyme to about 1g of tumor tissue, marking the sample with the name and the number of the sample, sealing a sealing film, digesting by using a shaker at 37 ℃ and 300rpm, and observing whether the digestion is finished every 1 h;
(4) after completion of the digestion, the mixture was centrifuged at 1400rpm for 5 minutes, the supernatant was discarded, the mixture was neutralized with an equal volume of a medium for cell separation containing 10% fetal bovine serum, then a 100 μm filter (manufacturer: Biosharp) was used to filter out undigested tissue masses, the tissue masses on the filter were washed with the medium for cell separation into a centrifuge tube (to reduce cell loss), and centrifuged at 1400rpm for 5 minutes;
(5) discarding the supernatant, observing whether there are blood cells, adding 10mL of blood cell lysate (manufacturer: Sigma) if there are blood cells, mixing, lysing at 4 ℃ for 20 minutes, reversing and mixing once, and then centrifuging at 1400rpm for 5 minutes;
(6) the supernatant was discarded, 10mL of cell separation medium was added to resuspend and collect the cells for expanded culture.
3. Culturing trophoblast cells
(1) NIH-3T3 (purchased from ATCC) was cultured in DMEM medium (manufacturer: Corning) supplemented with 10% FBS and 1% double antibody solution at 37 ℃ with 5% CO2Culturing under the condition;
(2) at passage, the medium was aspirated off and washed with PBS (phosphate buffered saline (1X), 0.0067M (PO)4) Washed once, digested with 0.25% pancreatin (available from Corning, usa) at 37 ℃ for 3 minutes, and neutralized with the same volume of DMEM medium supplemented with 10% FBS (available from Gibco, usa) and 1% double antibody solution when the cells were rounded and partially suspended to obtain a cell suspension;
(3) centrifuging the cell suspension at 1000rpm for 5 minutes, and removing the supernatant;
(4) cells were resuspended in DMEM supplemented with 10% FBS and 1% double antibody solution, plated at one-to-three ratio, and passaged every three days.
4. Irradiating trophoblast cells
(1) When NIH-3T3 grows to about 80% density, digesting the cells according to the method of 3(2) and resuspending in complete DMEM medium;
(2) irradiating by using gamma rays, wherein the irradiation dose is 25 Gy;
(3) the irradiated trophoblast cells are used in subsequent culture experiments.
Example 2 optimization of composition of Primary liver cancer cell culture Medium
A culture medium obtained by adding 1% double antibody solution (purchased from Corning Corp., wherein the concentration of streptomycin in the mother liquor is 10mg/ml and the concentration of penicillin is 10000U/ml), 10% fetal bovine serum (purchased from Gibco Corp.), and 0.1% Primocin solution (purchased from Invivogen, wherein the concentration of Primocin in the mother liquor is 50mg/ml) to DMEM/F12 medium was used as a basal medium.
Cytokines were added to the basal medium at the concentrations shown in table 1 below to obtain a single factor-supplemented medium of example 2.
Primary liver cancer cells were obtained from the excised liver cancer tissue samples of 4 patients (patient 2, patient 3, patient 7, and patient 8) with the same specifications and consent according to the method described in example 1. The primary liver cancer cells obtained as described above were cultured in viable cell density of 1X 10 using the single factor-supplemented medium and basal medium of example 2, respectively4Per cm2Seeded in 12-well plates (4.5 ten thousand cells per well) at a cell density of 5X 104Per cm2NIH-3T3 cells irradiated by gamma ray (irradiation dose 25Gy) were added and mixed well. Sterilizing the surface, and placing at 37 deg.C and 5% CO2Incubators (purchased from Saimeri fly) for culture.
The culture was carried out until day 8, the 12-well plate was removed, rinsed for 1 minute with 200. mu.l of 0.25% trypsin (from Gibco), aspirated, and 500. mu.l of 0.05% trypsin (from Gibco) was added to each well, and the mixture was incubated at 37 ℃ and 5% CO2The reaction was carried out in an incubator for 10 minutes until the cells were completely digested under a microscope (EVOS M500, Invitrogen), and after centrifugation at 1500rpm for 4 minutes, the supernatant was discarded, 1 ml of a basal medium was added for resuspension, and the cells were counted using a flow cytometer (JIMBIO FIL, Jiangsu microbial science, Ltd.) to obtain the total number of cells.
When at least three of 4 primary liver cancer cells had the effect of promoting proliferation when the medium added with the additive was used, compared with the basal medium, the medium was marked as "+"; when at least two of the 4 primary liver cancer cells showed the effect of suppressing proliferation when the medium containing the additive was used as compared with the case of using the basal medium, "-".
The results are shown in Table 1.
[ TABLE 1 ]
Figure BDA0002405588660000091
As shown in Table 1, Glu (glutamine), NEAA (non-essential amino acid), bFGF (basic fibroblast growth factor), HGF (hepatocyte growth factor), IL-6, hydrocortisone, EGF (epidermal growth factor), Insulin, Y27632, N2 additive, B27 additive and R-spondin1 all have strong proliferation effect on primary liver cancer cells.
Example 3 culture and characterization of Primary hepatocarcinoma cells
The primary liver cancer cell culture medium (HCCM) of example 3 was prepared according to the formulation of table 2 below.
[ TABLE 2 ]
Serial number Media additive species Suppliers of goods Final concentration
1 N2 Gibico 1:100
2 Epidermal growth factor R&D 10ng/ml
3 Hepatocyte growth factor Sino Biological 20ng/ml
4 Basic fibroblast growth factor Sino Biological 20ng/ml
5 R-spondin1 Sino Biological 250ng/ml
6 Glutamine Invitrogen 250ng/ml
7 Non-essential amino acids Corning 1μM
8 Insulin Sigma-Aldrich 5ng/ml
9 Y27632 MCE 10μM
10 IL-6 R&D 5ng/ml
11 DMEM/F12 medium Corning 90% (volume%)
12 Fetal bovine serum Gibico 10% (volume%)
13 Hydrocortisone Sigma-Aldrich 400ng/ml
14 Streptomycin-penicillin Corning 1% (volume%)
15 Primocin Invivogen 0.1% (volume%)
16 B27 Gibco 1:50
Primary liver cancer cells were obtained from a surgically excised liver cancer tissue sample of 1 patient with liver cancer (patient 1) who had been described and who had obtained consent, according to the method described in example 1. Using the primary liver cancer cell culture medium of example 3, the primary liver cancer cells obtained above were cultured at a viable cell density of 1X 104Per cm2Inoculating in 12-well plates (per well)4.5 ten thousand cells), at a cell density of 5X 104Per cm2NIH-3T3 cells irradiated by gamma ray (irradiation dose 25Gy) were added and mixed well. Sterilizing the surface, and placing at 37 deg.C and 5% CO2Incubators (purchased from Saimeri fly) for culture.
The observation under the mirror was performed on the third and seventh days of culture, respectively.
Fig. 1 (a) is an under-lens photograph (Invitrogen EVOS M500, 10-fold objective lens) of the culture to the third day, and fig. 1 (B) is an under-lens photograph (Invitrogen EVOS M500, 10-fold objective lens) of the culture to the seventh day. The circles of FIG. 1 (A) and (B) circle out the formed cell clones, and as shown in FIG. 1 (A), the cultured primary liver cancer cell can form typical cell clones within 3 days, and as shown in FIG. 1 (B), the cell clones are rapidly proliferated.
On the 28 th day of culture, immunohistochemical analysis was performed on the liver cancer cells obtained by the culture. The immunohistochemical method is as follows.
Digesting and centrifuging the cultured primary liver cancer cells, washing the cells for 1-2 times by PBS (purchased from Shanghai Producers), then fixing the cells in 95% ethanol (purchased from Shanghai Producers), centrifuging the fixed cells, removing supernatant, wrapping cell precipitates by embedding paper (purchased from Shanghai Producers), and soaking the cell precipitates in 95% ethanol again for preparing cell wax blocks. Samples were prepared for cellular wax blocks, followed by analysis of liver cancer immunohistochemical markers (Ki67, CK7, both from Cell Signaling Technology).
Paraffin sections were sequentially placed in xylene 1 (from shanghai bio-reactor) for 10 minutes, xylene 2 (from shanghai bio-reactor) for 10 minutes, 100% ethanol 1 (from shanghai bio-reactor) for 5 minutes, 100% ethanol 2 (from shanghai bio-reactor) for 5 minutes, 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes in a fume hood, and then washed 3 times with PBS, 5 minutes/time, and placed in a clear solution (PBS + 0.3% H + 0.3%) in a clear solution2O2+ 0.3% TritonX-100 (from Shanghai Producer) was incubated at 100rpm on a shaker for 30 min at room temperature in the dark and washed 3 times with PBS for 5 min/time. Then, the stained tissue was circled with an immunohistochemical pen, 5% goat serum or donkey serum (0.3% Triton X-100 in PBS diluted 1: 50) was dropped, 37 deg.CAnd (4) incubating in a water bath for 30 minutes (the II antibody is closed by sheep serum if the II antibody is sheep antibody, and is closed by donkey serum if the II antibody is donkey antibody). Antiserum was removed, and the I antibody was diluted 1:50 with 0.3% triton X-100 PBS (purchased from Shanghai, Ltd.), dropped into the sections, incubated in a 37 ℃ water bath for 1h, and transferred to a 4 ℃ refrigerator for overnight incubation. Tissue sections incubated with the I antibody were removed, equilibrated to room temperature, washed 3 times with PBS for 5 min/time. 0.3% triton X-100 in PBS was diluted 1:50 with I antibody (biotin), dropped into the tissue sections, incubated in a 37 ℃ water bath for 1h, washed 3 times with PBS, 5 minutes/time. Adding hematoxylin (purchased from Shanghai, China) dropwise, dyeing for 5-10 minutes at room temperature, and dyeing with ddH2And washing for 3 times by using O. Adding 0.5% ethanol hydrochloride (prepared from 70% ethanol), reacting for 30s, bluing with 0.5% ammonia water for 30s, and adding ddH2Wash 3 times O and take pictures in a microscope (EVOS M500, 10 x objective lens, Invitrogen).
The results are shown in FIG. 2. FIG. 2 (A) is the immunohistochemistry result of the cells cultured with the primary liver cancer cell culture medium of this example, and FIG. 2 (B) is the immunohistochemistry result of the liver cancer tissue of the same patient. FIGS. 2 (A) and (B) are shown as CK7(-), Ki67(+), that is, the liver cancer cells cultured by the primary liver cancer cell culture medium of this example can maintain the pathological characteristics of the liver cancer tissue.
Example 4 Primary culture period and cell count statistics for Primary hepatocarcinoma cells
In the same manner as in example 3, primary liver cancer cells were obtained from the excised liver cancer tissue samples of 9 patients (1 to 9) with consented liver cancer, which were described and obtained using the culture medium prepared in example 3, and were cultured in the same manner as in example 3 using the primary liver cancer cell culture medium of example 3. Digestion was performed when the cells had expanded to 85% and counted, and the number of days of culture until digestion was recorded as one culture cycle, with the results shown in table 3 below.
[ TABLE 3 ]
Sample information Days of culture Number of seeded cells (. about.10)4) Cell number (. 10) was collected4)
Patient 1 18 6 100
Patient 2 13 10 180
Patient 3 13 10 160
Patient 4 13 10 164
Patient 5 10 10 440
Patient 6 14 10 264
Patient 7 22 8 280
Patient 8 12 10 178
Patient 9 11 10 276
Mean value of 12.6 8.4 204
As shown in Table 3, the liver cancer cells cultured by using the primary liver cancer cell culture medium of the invention can be 10 days in 12.6 days on average4Of the order of 106Order of magnitude of proliferation.
Example 5 Effect of the concentration of the factor added on the proliferation of hepatoma cells
According to the method described in example 1, primary liver cancer cells were obtained from the liver cancer tissue samples obtained by surgical resection of 4 patients (patient 4, patient 8, patient 9, and patient 10) who had been described and given consent, and were cultured using the primary liver cancer cell culture medium described in example 3, and the obtained liver cancer cells were cultured at a viable cell density of 1 × 104Per cm2Seeded in 12-well plates (4.5 ten thousand cells per well) at a cell density of 5X 104Per cm2NIH-3T3 cells irradiated by gamma ray (irradiation dose 25Gy) were added and mixed well. Sterilizing the surface, and placing at 37 deg.C and 5% CO2Incubator (purchased from Saimeri fly) for cultivation. After the cells had expanded to 85%, the 12-well plate was removed, rinsed with 200. mu.l of 0.25% trypsin (from Gibco) for 1 minute, aspirated, and 500. mu.l of 0.05% trypsin (from Gibco) was added to each well, and the mixture was incubated at 37 ℃ and 5% CO2The reaction was carried out in an incubator for 10 minutes until the cells were completely digested under a microscope (EVOS M500, Invitrogen), and after centrifugation at 1500rpm for 4 minutes, the supernatant was discarded, 1 ml of a basal medium was added for resuspension, and the cells were counted using a flow cytometer (JIMBIO FIL, Jiangsu microbial science, Ltd.) to obtain the total number of cells. The cells obtained were used in the following culture experiments.
Next, 12 media formulations were prepared for the experiments:
formula 1: the primary liver cancer cell culture medium in example 3 contains no N2 additive;
and (2) formula: the culture medium component of the primary liver cancer cells in example 3 does not contain non-essential amino acids;
and (3) formula: the primary liver cancer cell culture medium component in example 3 does not contain glutamine;
and (4) formula: the primary liver cancer cell culture medium component of example 3 does not contain insulin;
and (5) formula: the primary liver cancer cell culture medium in example 3 contains no B27 additive;
and (6) formula: HGF is not contained in the culture medium components of the primary hepatoma cells in example 3;
and (3) formula 7: the primary liver cancer cell culture medium in example 3 contains no Y27632;
and (4) formula 8: the primary liver cancer cell culture medium component in example 3 does not contain hydrocortisone;
formula 9: the primary liver cancer cell culture medium component in example 3 does not contain EGF;
formula 10: the primary liver cancer cell culture medium component in example 3 does not contain bFGF;
formula 11: the culture medium component of the primary liver cancer cells in example 3 does not contain R-spondin 1;
formula 12: the culture medium component of the primary liver cancer cells in the example 3 does not contain IL-6;
the primary liver cancer cell culture media in the formulas 1-12 and the example 3 are respectively used for diluting the digested cell suspension, and the cell suspension is planted into a 24-well plate according to the volume of 500 microliters of 2 ten thousand cells per well.
When the culture medium of the formula 1 is used, 5 concentration gradients of N2 additives are prepared according to final concentrations of 1:400, 1:200, 1:100, 1:50 and 1:25 respectively, and 500 microliters of the prepared N2 additive is added into a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 1.
When the culture medium of formula 2 is used, 5 non-essential amino acids with concentration gradients are prepared according to final concentrations of 4 μ M, 2 μ M, 1 μ M, 0.5 μ M and 0.25 μ M respectively, and 500 microliters of the prepared non-essential amino acids are added into a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 2.
When the culture medium of formula 3 is used, 5 glutamine concentration gradients are prepared according to final concentrations of 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL and 62.5ng/mL respectively, and 500 microliters of prepared glutamine per well is added to a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 3.
When the culture medium of formula 4 is used, 5 concentration gradients of insulin are prepared according to final concentrations of 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL and 1.25ng/mL respectively, and 500 microliters of prepared insulin per well is added to a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 4.
When the culture medium of formula 5 is used, 5 concentration gradients of B27 additives are prepared according to final concentrations of 1:200, 1:100, 1:50, 1:25 and 1:12.5 respectively, and 500 microliters of the prepared B27 additives are added into a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 5.
When the culture medium of formula 6 is used, 5 HGF with concentration gradients of 80ng/mL, 40ng/mL, 20ng/mL, 10ng/mL and 5ng/mL are prepared according to the final concentration respectively, and 500 microliters of prepared HGF is added into a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 6.
When the culture medium of formula 7 is used, 5 concentration gradients of Y27632 are prepared according to final concentrations of 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M and 2.5. mu.M, and 500. mu.L of prepared Y27632 per well is added to a 24-well plate inoculated with primary cells; and control wells (BC) were set using medium of formula 7.
When the culture medium of formula 8 is used, 5 concentration gradients of hydrocortisone are prepared according to final concentrations of 1.6. mu.g/mL, 0.8. mu.g/mL, 0.4. mu.g/mL, 0.2. mu.g/mL and 0.1. mu.g/mL respectively, and 500. mu.L of the prepared hydrocortisone per well is added to a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 8.
When the culture medium of formula 9 is used, EGF with 5 concentration gradients is prepared according to final concentrations of 40ng/mL, 20ng/mL, 10ng/mL, 5ng/mL and 2.5ng/mL respectively, and 500 microliter of the prepared EGF is added to each well of a 24-well plate inoculated with primary cells; and control wells (BC) were set using medium of formula 9.
When the culture medium of the formula 10 is used, 5 bFGF with concentration gradients are prepared according to final concentrations of 80ng/mL, 40ng/mL, 20ng/mL, 10ng/mL and 5ng/mL respectively, and 500 microliters of prepared bFGF is added into a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 10.
When the culture medium of the formula 11 is used, 5 concentration gradients of R-spondin1 are prepared according to the final concentrations of 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL and 62.5ng/mL respectively, and 500 microliters of prepared R-spondin1 is added into a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 11.
When the culture medium of formula 12 is used, 5 concentration gradients of IL-6 are prepared according to final concentrations of 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL and 1.25ng/mL, and 500 microliters of prepared IL-6 is added to a 24-well plate inoculated with primary cells; and control wells (BC) were set using medium of formula 12.
Simultaneously, 1 ten thousand irradiated NIH-3T3 cells were added to each well as trophoblasts.
When the cells were expanded to about 85% of the 24 wells and digested, the ratio was calculated with reference to the number of cells in the control well (BC), and the results are shown in FIGS. 3 (A) to (L), respectively. In fig. 3 (a) to (L), the ratio is the ratio of the number of cells obtained by one-pass culture using each medium to the number of cells obtained by one-pass culture using the corresponding control well. The ratio is more than 1, which indicates that the proliferation promoting effect of the prepared culture medium containing different concentration factors or small molecular compounds is better than that of the culture medium of the control hole, and the ratio is less than 1, which indicates that the proliferation promoting effect of the prepared culture medium containing different concentration factors or small molecular compounds is weaker than that of the culture medium of the control hole.
As shown in fig. 3 (a) to (L), the concentration range of the N2 additive is 1:400 to 1:25 in terms of volume ratio, and the cell proliferation effect is most remarkable when the additive is added at 1: 100; the concentration range of the non-essential amino acid is 0.25-4 mu M, and the cell proliferation effect is most obvious when the concentration is 1 mu M; the concentration range of glutamine is 62.5-1000 ng/mL, and the cell proliferation effect is most obvious when the concentration is 250 ng/mL; the concentration range of insulin is 1.25-20 ng/mL, and the cell proliferation effect is most obvious when the concentration is 5-20 ng/mL; the concentration range of the B27 additive is 1: 12.5-1: 200 in terms of volume ratio, and the cell proliferation effect is most obvious when the B27 additive is added in a ratio of 1: 12.5-1: 50; the HGF concentration range is 5-80 ng/mL, and the cell proliferation effect is most obvious when the concentration is 20-80 ng/mL; the concentration range of Y27632 is 5-40 mu M, and the cell proliferation effect is most obvious when the concentration is 10 mu M; the concentration range of the hydrocortisone is 0.4-1.6 mu g/mL, and the cell proliferation effect is most obvious when the concentration is 0.4 mu g/mL; the concentration range of EGF is 2.5-40 ng/mL, and the cell proliferation effect is most obvious when the concentration is 10 ng/mL; the bFGF concentration range is 5-80 ng/mL, and the cell proliferation effect is most obvious when the bFGF concentration range is 20-80 ng/mL; the concentration range of R-spondin1 is 62.5-1000 ng/mL, and the cell proliferation effect is most obvious when the concentration is 250 ng/mL; the concentration range of IL-6 is 1.25-20 ng/mL, and the cell proliferation effect is most obvious when the concentration is 5-20 ng/mL.
Example 6 comparison with existing Medium
FM medium was formulated according to the formulation of table 4 below.
[ TABLE 4 ]
Media composition Suppliers of goods Final concentration
DMEM medium Corning 65% (volume%)
Fetal bovine serum Gibico 10% (volume%)
Ham's F12 nutrient solution Gibico 25％
Hydrocortisone Sigma-Aldrich 25ng/ml
Epidermal growth factor R&D 0.125ng/ml
Insulin Sigma-Aldrich 5μg/ml
Amphotericin B Sigma-Aldrich 250ng/ml
Gentamicin Gibico 10μg/ml
Cholera toxin Sigma-Aldrich 0.1nM
Y27632 Enzo 10μM
Primary liver cancer cells obtained by surgical resection of liver cancer tissue samples from the described and approved liver cancer patients (patient 3 and patient 4) were cultured in the same manner as in example 3 using the FM medium and the primary liver cancer cell culture medium (HCCM in the figure) in example 3.
On day 3 of culture, observation was performed using an inverted microscope (EVOS M500, 10-fold, Invitrogen) and the culture results of primary liver cancer cells from patient 3 are shown in fig. 4. FIG. 4 (A) shows primary hepatocarcinoma cells cultured using FM medium, FIG. 4 (B) shows primary hepatocarcinoma cells cultured using the primary hepatocarcinoma cell culture medium of example 3, and as shown in FIG. 4, when the primary hepatocarcinoma cell culture medium of example 3 was used, significant cell clone was formed on day 3, and the effect was superior to that of FM medium.
The culturing was continued, and the growth curves of two primary liver cancer cells cultured using two different media were plotted using Graphpad prism7.0 software with the number of days of culturing on the abscissa and the number of cell population doublings on the ordinate, and the culture results of primary liver cancer cells from patient 3 and patient 4 are shown in fig. 5 (a) and fig. 5 (B), respectively. From (a) and (B) of fig. 5, it was confirmed that the proliferation rate of primary liver cancer cells was superior to that of FM medium when the primary liver cancer cell culture medium of example 3 was used.
Example 7 xenograft tumor formation experiment of primary liver cancer cells cultured in the inventive primary liver cancer cell culture Medium in mice
Primary liver cancer cells were obtained from a sample of a surgically excised liver cancer tissue of a patient with liver cancer (patient 4) who had been described and given consent, and cultured in the same manner as in example 3 using the primary liver cancer culture medium of example 3 until the number of liver cancer tumor cells reached 1X 107In one occasion, the liver cancer cells are digested by the digestion method in example 2, collected, counted, mixed well by the primary liver cancer cell culture medium of example 3, and 5 × 10 μ L is aspirated6Each hepatoma carcinoma tumor cell was resuspended, injected into the subcutaneous site of a 6-week-old female hyperimmune deficient (NCG) mouse (purchased from Nanjing model animal institute), and the tumor formation volume and growth rate of hepatoma carcinoma cells in the mouse were observed every three days and recorded.
FIG. 6 is a record of tumor volumes in mice of hepatoma cells cultured from primary hepatoma cells from patient 4.
As shown in fig. 6, tumor formation was observed at the tumor cell inoculation site of the mouse on day 13 after tumor cell inoculation, and tumor proliferation in the mouse was significant from day 13 to day 22, which indicates that the liver cancer tumor cells derived from the liver cancer tissue cultured by the primary liver cancer cell culture medium and the culture method of example 3 had tumorigenicity in the mouse.
Example 8 drug sensitivity test of liver cancer cells derived from liver cancer tissue
Taking the surgical resection sample of the liver cancer patient as an example, the liver cancer cells cultured from the liver cancer sample of the patient can be used for detecting the sensitivity of the liver cancer cells of the patient to different drugs.
1. Plating primary liver cancer cells: primary liver cancer cells were obtained from the excised liver cancer tissue samples of 4 patients (patient 1 to patient 4) with the same consent as described in example 1, and used in the method described in example 3The primary liver cancer culture medium is cultured in the same way as in example 3 until the number of liver cancer cells reaches 1X 107In one occasion, the digestion method in the embodiment 2 is adopted to digest the liver cancer cells, and the liver cancer cells are collected, counted and inoculated into a 384-well plate according to the density of 3000-5000 cells/well so that the cells adhere to the wall overnight.
2. Drug gradient experiments:
(1) preparing a drug storage plate by adopting a concentration gradient dilution method: respectively sucking 10 μ L of the mother liquid (concentration of the mother liquid is 2 times of the maximum blood concentration C of the drug in human body)maxFormulation), added to a 0.5mL EP tube containing 20. mu.L of DMSO, and then 10. mu.L of the solution was pipetted from the above EP tube into a second 0.5mL EP tube already containing 20. mu.L of DMSO, i.e., the drug was diluted 1: 3. Repeating the above method, sequentially diluting to obtain 7 concentrations required by dosing. Drugs at different concentrations were added to 384-well drug storage plates. Solvent control an equal volume of DMSO was added to each well as a control. In this example, the drugs to be tested were sorafenib (MCE), regorafenib (MCE), 5-fluorouracil (MCE) and doxorubicin (MCE).
(2) Different concentrations of drug and solvent controls in 384-well drug storage plates were added to the hepatoma-plated 384-well cell culture plates obtained in step 1 above using a high throughput automated workstation (available from Perkin Elmer), each with 3 replicates of the drug and solvent controls. The volume of drug added per well was 100 nL.
(3) And (3) detecting the activity of the cells: after 72 hours of administration, the chemiluminescence values of the cells after the drug addition culture are detected by using a Cell Titer-Glo detection reagent (manufactured by Promega), the chemiluminescence values reflect the Cell viability and the influence of the drug on the Cell viability, the prepared Cell Titer-Glo detection solution is added into each hole, and the chemiluminescence values are detected by using an enzyme-linked immunosorbent assay (ELISA) instrument after the mixture is uniformly mixed.
The median inhibition IC50 was plotted and calculated using Graphpad Prism7.0 software.
(4) The results of the drug susceptibility testing are shown in figure 7.
Fig. 7 (a) to (D) show drug sensitivities of hepatoma tumor cells cultured from surgically excised cancer tissue samples of four different hepatoma patients to the two chemotherapeutic drugs doxorubicin and 5-fluorouracil, and to the targeted drugs sorafenib and regorafenib, respectively. The results show that cells from the same patient have different sensitivities to different drugs, and that cells from different patients also have different sensitivities to the same drug.
According to fig. 7, it can be confirmed that the sensitivity test result of the liver cancer cells cultured by the technology of the invention from the cancer tissues of the liver cancer patient to the chemotherapeutic drugs and the targeted drugs is consistent with the clinical medication remission rate of the patient, for example, the chemotherapeutic drugs (doxorubicin) show wide drug resistance, while the first-line clinical targeted drug of the liver cancer, sorafenib, has effectiveness in only part of patients, and the application potential of the liver cancer cells cultured by the technology of the invention in predicting the clinical medication curative effect of the liver cancer patient is suggested.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (9)

1. A primary liver cancer cell culture medium for culturing primary liver cancer cells is characterized in that:
in a primary medium comprising glutamine, non-essential amino acids, basic fibroblast growth factor, hepatocyte growth factor, IL-6, epidermal growth factor, insulin, Y27632, N2 additive, B27 additive, Primocin, penicillin and streptomycin, fetal calf serum, and optionally hydrocortisone, optionally R-spondin1,
the initial culture medium is selected from DMEM/F12, DMEM, F12 or RPMI-1640.
2. Primary liver cancer cell culture medium according to claim 1, characterized in that:
the content of the glutamine is 62.5 ng/mL-1000 ng/mL;
the non-essential amino acid is one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the total content of the non-essential amino acid is 0.25-4 mu M;
the content of the basic fibroblast growth factor is 5ng/ml to 80 ng/ml;
the content of the hepatocyte growth factor is 5ng/ml to 80 ng/ml;
the content of the IL-6 is 1.25ng/ml to 20 ng/ml;
the content of the epidermal growth factor is 2.5 ng/ml-40 ng/ml;
the content of the insulin is 1.25 ng/ml-20 ng/ml;
the content of the Y27632 is 5-40 mu M;
the volume ratio of the N2 additive to the culture medium is 1: 25-1: 400;
the volume ratio of the B27 additive to the culture medium is 1: 12.5-1: 200;
the content of the Primocin is 40 to 80 mu g/ml;
the content of the penicillin is 80U/ml-200U/ml;
the streptomycin content is 50-150 mug/ml;
the volume ratio of the fetal calf serum to the culture medium is 1: 5-1: 20;
if the hydrocortisone is contained, the content of the hydrocortisone is 0.4 mu g/ml-1.6 mu g/ml;
if the R-spondin1 is contained, the content is 62.5 ng/mL-1000 ng/mL.
3. A method for culturing primary liver cancer cells is characterized by comprising the following steps:
culturing primary liver cancer cells using the primary liver cancer cell culture medium of claim 1 or 2.
4. The culture method according to claim 3, wherein:
in the culture, 5X 104～1.5×105Per cm2The irradiated trophoblast cells are added to the cell density of (a).
5. The culture method according to claim 4, wherein:
the irradiated trophoblast cell is a mouse fibroblast, preferably NIH-3T3 or J2-3T 3.
6. The culture method according to claim 4, wherein:
the irradiated trophoblast cells are irradiated with gamma rays or X-rays, preferably gamma rays.
7. The culture method according to claim 6, wherein:
the irradiation dose is 10-50 Gy.
8. The culture method according to any one of claims 3 to 7, wherein:
and (3) carrying out digestion passage when the primary liver cancer cells grow to be more than 75% of the bottom area of the culture vessel.
9. A method of assessing the efficacy of a drug for the treatment of a liver cancer disease, comprising the steps of:
(1) culturing the liver cancer cell according to the culture method of any one of claims 3-8;
(2) selecting the drug to be tested at its maximum plasma concentration CmaxFor reference, 2-5 times of CmaxDiluting to different drug concentration gradients for the initial concentration;
(3) digesting the liver cancer cells cultured in the step (1) into a single cell suspension, adding the single cell suspension into a perforated plate for adherence, and adding the drug after gradient dilution to the adherence cells;
(4) and (5) carrying out cell viability detection.
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