CN117757749A - Culture medium and culture method of kidney cancer primary cells - Google Patents

Culture medium and culture method of kidney cancer primary cells Download PDF

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CN117757749A
CN117757749A CN202410132897.7A CN202410132897A CN117757749A CN 117757749 A CN117757749 A CN 117757749A CN 202410132897 A CN202410132897 A CN 202410132897A CN 117757749 A CN117757749 A CN 117757749A
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culture medium
primary
concentration range
kidney cancer
cells
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汪悦
李跃兵
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Mirror Image Qidian Shanghai Cell Technology Co ltd
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Mirror Image Qidian Shanghai Cell Technology Co ltd
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Abstract

The invention discloses a medium and a culture method of kidney cancer primary cells, wherein the medium consists of an initial medium, a ROCK inhibitor, nicotinamide, antibiotics, insulin, nonessential amino acid, B27, glutamine, fetal bovine serum and specific additive factors. The culture medium and the culture method of the primary kidney cancer cells can improve the success rate of primary kidney cancer cell culture, and the success rate reaches more than 90 percent; ensuring that the kidney cancer cells which are primarily cultured in vitro can reproduce pathological phenotype and heterogeneity of primary cell-derived patients; the cultured primary renal cancer cells are not interfered by mesenchymal cells such as fibroblasts, adipocytes and the like, and purified renal cancer cells can be obtained; the efficiency of amplifying the primary renal cancer cells is high, and only 10 4 The cell number of the grade can be amplified to 10 in about two weeks 6 The order of magnitude liver cancer cells, and the amplified liver cancer cells can be continuously passaged.

Description

Culture medium and culture method of kidney cancer primary cells
Technical Field
The invention relates to the technical field of biology, in particular to a culture medium and a culture method of kidney cancer primary cells.
Background
Renal cancer is a malignant tumor that originates in the renal parenchymal urinary tract epithelial system, one of the most common urinary system tumors. Depending on the type of pathology, kidney cancer mainly includes three subtypes: renal clear cell carcinoma (Clear cell renal cell carcinoma, ccRCC), renal papillary carcinoma (Papillary renal cell carcinoma, pRCC), and renal chromocytocarcinoma (Chromophobe renal cell carcinoma, crRCC).
The existing in vitro cultured kidney cancer cell line is mainly obtained by spontaneously immortalizing normal cells by long-term culture or transfecting oncogenes which promote the immortalization of the normal cells. Cell lines established by traditional methods remain the main mainstay of research in cell, molecular and cancer biology. However, these methods change the genetic background of cells, and long-term cultured cell lines are also prone to genomic instability, which can lead to artificial alterations in the phenotype of tumor cell lines and tumor cells in vivo. The complex heterogeneity of primary tumors is often lacking in these cell lines, limiting the application of these cell lines to predicting tumor cell responses, affecting the accuracy of scientific research and drug development for gastric cancer. In addition, in the process of culturing cells obtained from kidney cancer tissues into cancer cells, the conventional culture method is difficult to obtain the cancer cells, the problem that the cancer cells are easily interfered by fibroblasts in the culture process, the formed clones cannot be passaged and the like exists, and the application of primary cells of human kidney cancer is limited.
Disclosure of Invention
The invention provides a culture medium and a culture method of kidney cancer primary cells, and aims to solve the problems in the prior art.
The technical scheme provided by the invention is as follows:
in one aspect, the invention provides a kidney cancer primary cell culture medium, which is composed of a primary culture medium, a ROCK inhibitor, nicotinamide, antibiotics, insulin, non-essential amino acids, B27, glutamine, fetal bovine serum, and specific additive factors;
the initial culture medium is selected from GGT551, DMEM/F12 and RPMI-1640 culture medium;
the ROCK inhibitor is Y-27632, and the concentration range of the Y-27632 is 5-20 uM;
the concentration range of the nicotinamide is 5-40 uM;
the antibiotics comprise penicillin and streptomycin, and the concentration ranges of the penicillin and the streptomycin are 20-200 mug/mL;
the concentration range of the insulin is 2-30 mug/mL;
the total concentration range of the nonessential amino acids is 30-400 mu M;
the volume ratio of the B27 relative to the culture medium is 1:10-1:200;
the concentration range of the glutamine is 0.1-10 mM;
the volume ratio of the fetal bovine serum to the culture medium is 1:5-1:20;
the concentration range of the specific additive factors is 10-100 ng/ml.
Further, the concentration range of the Y-27632 is 5-10 uM;
the concentration range of the nicotinamide is 10-20 uM;
the concentration ranges of the penicillin and the streptomycin are 50-100 mug/mL;
the concentration range of the insulin is 10-20 mug/mL;
the total concentration range of the nonessential amino acids is 100-300 uM;
the volume ratio of the B27 relative to the culture medium is 1:20-1:100;
the concentration of the glutamine is 1-5 mM;
the volume ratio of the fetal bovine serum to the culture medium is 1:5-1:10;
the concentration range of the specific additive factors is 30-70 ng/ml.
Further, the specific additive factors comprise a fibroblast growth factor (FGF-10) and a fibroblast growth factor 9 (FGF-9), and the volume ratio of the fibroblast growth factor (FGF-10) to the fibroblast growth factor 9 (FGF-9) is 1:10-1:20.
Further, the non-essential amino acids comprise glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine.
In another aspect, the present invention provides a method for culturing primary cells of renal cancer, wherein the primary cells of gastric cancer are cultured using the medium for primary cells of renal cancer described above.
Further, in the culture, the cell density is 1 to 4X 10 4 Individual/cm 2 The trophoblasts are added.
Further, the trophoblast is an irradiated NIH-3T3 cell, the irradiation source is X-ray or gamma-ray, and the irradiation dose is 20-30 Gy.
Compared with the prior art, the invention has the beneficial effects that:
the beneficial effects of the invention include:
(1) The success rate of primary kidney cancer cell culture is improved, and the success rate reaches more than 90 percent;
(2) Ensuring that the kidney cancer cells which are primarily cultured in vitro can reproduce pathological phenotype and heterogeneity of primary cell-derived patients;
(3) The cultured primary renal cancer cells are not interfered by mesenchymal cells such as fibroblasts, adipocytes and the like, and purified renal cancer cells can be obtained;
(4) The efficiency of amplifying the primary renal cancer cells is high, and only 10 4 The cell number of the grade can be amplified to 10 in about two weeks 6 The order of magnitude liver cancer cells, and the amplified liver cancer cells can be continuously passaged.
Drawings
FIG. 1 is a microscopic photograph of primary kidney cancer cells isolated from a kidney cancer tissue sample, which were obtained by culturing the primary kidney cancer cells using the primary cell culture medium of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. It will be apparent that the embodiments described below are some, but not all, of the embodiments of the present application.
EXAMPLE 1 acquisition of Primary renal cancer cells and treatment of trophoblast cells
1. Collection of renal cancer tissue samples
Renal cancer tissue samples were collected within half an hour after surgical excision or puncture of the patient who had been explained and obtained consent. Cutting non-necrotic parts under aseptic conditionRenal cancer tissue sample 1cm 3 The above was placed in a 20ml dmem/F12 medium with pre-chilled in a refrigerator at 4 ℃ in advance, and marked. When isolating the renal cancer tissue samples, regions with significant necrosis and non-tumor should be avoided.
2. Treatment of renal cancer tissue samples
(1) Tissue was taken in a biosafety cabinet and placed in a 100mm dish (Corning, 430167), the blood-bearing tissue was removed, a 1% streptomycin-penicillin solution (available from Corning, usa, with 50ug/ml streptomycin concentration in stock solution and 100ug/ml penicillin concentration) was added to DMEM/F12 medium (manufacturer: corning), and 1% ggt 551-derived medium (hereinafter referred to as cell separation medium) was washed 3 times, transferred to a new 100mm dish, 10ml cell separation medium was added, and mechanical separation was performed using a sterile scalpel, surgical scissors and forceps to divide the tissue into small pieces with a diameter of less than 1 mm;
(2) Transferring the minced tissue into a 50mL centrifuge tube, adding 5mL of culture medium for cell separation, uniformly mixing, and centrifuging at 1400rpm for 5 minutes;
(3) Carefully removing supernatant in the centrifuge tube by using a pipette, adding a cell separation culture medium and a tissue digestion solution prepared by 2X digestive enzymes (containing collagenase II (1 mg/mL) (manufacturer Sigma) and collagenase IV (1 mg/mL) (manufacturer Sigma)) according to the volume ratio of 1:1, uniformly mixing and re-suspending, wherein the added amount of the digestive enzymes is about 10mL of digestive enzymes used for 1g of tumor tissue, labeling the sample with the sample name and number, sealing by a sealing film, and performing shaking table digestion at 37 ℃ and 300rpm, and observing whether digestion is completed every 1 h;
(4) After digestion is completed, centrifuging at 1400rpm for 5 minutes, discarding supernatant, neutralizing with an equal volume of cell separation medium containing 10% fetal bovine serum, filtering undigested tissue mass with a 100 μm filter screen (manufacturer: biosharp), washing the tissue mass on the filter screen into a centrifuge tube with the cell separation medium (reducing cell loss), and centrifuging at 1400rpm for 5 minutes;
(5) Discarding the supernatant, observing whether blood cells exist, if so, adding 10mL of blood cell lysate (manufacturer: sigma), mixing, cracking for 20 minutes at 4 ℃, reversing the mixing for one time, and centrifuging at 1400rpm for 5 minutes;
(6) The supernatant was discarded, 10mL of the cell separation medium was added to resuspend and the cells were collected for expansion culture.
3. Culturing trophoblast cells
(1) NIH-3T3 (from ATCC) was cultured in DMEM medium (manufacturer: corning) supplemented with 10% FBS, 1% streptomycin-penicillin solution and 1% GGT551 (TAKARA brand) at 37℃and 5% CO 2 Culturing under the condition;
(2) At passage, the medium was aspirated off, and PBS (phosphate buffer (1X), 0.0067M (PO) 4 ) Washing once, performing first digestion treatment in inhibitor containing type II collagenase to obtain first digestion product, placing the first digestion product in pancreatin substitute containing inhibitor to perform second digestion treatment to obtain second digestion product, and neutralizing with the same volume of DMEM medium added with 10% FBS (purchased from Gibco company of America) and 1% streptomycin-penicillin solution when cells become round and are partially suspended to obtain cell suspension; the concentration of type II collagenase is 5mg/ml, the inhibitor is ROCK inhibitor Y-27632, the concentration of the inhibitor is 10 mu M, the pancreatin substitute is TrypLE Express, and the concentration is 5 mu M.
(3) Centrifuging the cell suspension at 1000rpm for 5 minutes, and discarding the supernatant;
(4) Cells were resuspended in DMEM supplemented with 10% fbs, 1% streptomycin-penicillin solution and 1% ggt551, plated in a one-to-three ratio and passaged every three days.
4. Irradiation of trophoblast cells
(1) When NIH-3T3 grows to about 80% of density, digesting the cells according to the method of 3 (2), and re-suspending the cells in a complete DMEM medium;
(2) Gamma rays are used for irradiation, and the irradiation dose is 25Gy;
(3) The irradiated trophoblast cells were used in subsequent culture experiments.
Example 2 optimization of the composition of the Primary renal cancer cell Medium
A medium obtained by adding 1% streptomycin-penicillin solution, 1% GGT551, and 10% fetal bovine serum (available from Gibco corporation) 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, respectively, to obtain a single factor-added medium of example 2.
Primary renal cancer cells were obtained from surgical excision of renal cancer tissue samples from 4 patients who were explained and obtained as consent according to the method described in example 1. The primary renal cancer cells obtained as described above were cultured in a viable cell density of 2X 10 using the single factor addition medium and the basal medium of example 2, respectively 4 Individual/cm 2 Inoculated in 12-well plates (4.5 ten thousand cells per well) according to a cell density of 4X 10 4 Adding NIH-3T3 cells irradiated by gamma rays (irradiation dose 25 Gy) into each cm, and uniformly mixing. Sterilizing the surface, placing at 37deg.C and 5% CO 2 Incubator (purchased from zemoeid) culture.
The culture was carried out until day 7, the 12-well plate was removed, rinsed with 200. Mu.l of 0.25% trypsin (from Gibco Co.) for 1 minute, and after blotting, 500. Mu.l of 0.05% trypsin (from Gibco Co.) was added to each well, and the wells were placed at 37℃and 5% CO 2 The reaction was carried out in an incubator for 10 minutes until complete digestion was observed under a microscope (Invitrogen company EVOS M500), and after centrifugation at 1500rpm for 4 minutes, the supernatant was discarded, 1 ml of basal medium was added for resuspension, and the total number of cells was counted using a flow image counter.
When at least three of 4 cases of primary renal cancer cells have proliferation promoting effects in the case of using the medium to which the additive is added, as compared with the case of using the basal medium, it is denoted as "+"; at least two cases among 4 cases of primary renal cancer cells when the medium to which the additive was added showed an effect of inhibiting proliferation, as compared with when the basal medium was used, were denoted as "-".
The experimental results are shown in table 1.
TABLE 1
Based on the above results, further culture experiments were performed with insulin, Y-27632, fibroblast growth factor (FGF-10), fibroblast growth factor 9 (FGF-9), non-essential amino acids, B27, nicotinamide, and other factors.
Example 2 Effect of combinations of different additive factors in Primary Medium for Kidney cancer on proliferation of Primary cells for Kidney cancer
The kidney cancer primary cell culture medium of different additive factor combinations was prepared according to the ingredients in table 2, and the proliferation promoting effect of the different additive factor combinations on the kidney cancer primary cells was examined.
TABLE 2 preparation of different component Medium (final concentration)
Obtaining kidney cancer primary cells from kidney cancer tissue according to the method of steps (2) to 3 of example 1, dividing the obtained cell suspension into 8 parts on average, centrifuging at 1500rpm for 4 minutes, re-suspending with 200. Mu.l BM, 1# to 7# medium, respectively, after centrifugation, and obtaining a viable cell density of 2X 10, respectively 4 Individual/cm 2 Inoculated in 12-well plates (4.5 ten thousand cells per well) according to a cell density of 4X 10 4 Adding NIH-3T3 cells irradiated by gamma rays (irradiation dose 25 Gy) into each cm, and uniformly mixing. Sterilizing the surface, placing at 37deg.C and 5% CO 2 Incubator (purchased from zemoeid) culture.
On day 7 of incubation, the 12-well plate was removed, rinsed with 200. Mu.l of 0.25% trypsin (from Gibco) for 1 minute, blotted off, and then 500. Mu.l of 0.05% trypsin (from Gibco) were added to each well, and placed at 37℃and 5% CO 2 The reaction was carried out in an incubator for 10 minutes until complete digestion was observed under a microscope (Invitrogen company EVOS M500), and after centrifugation at 1500rpm for 4 minutes, the supernatant was discarded, 1 ml of basal medium was added for resuspension, and the total number of cells was counted using a flow image counter. Will be divided intoThe results obtained from primary cells of renal cancer isolated from endoscopic tissue samples are shown in fig. 1.
As can be seen from the results of FIG. 1, the proliferation of primary gastric cancer cells was promoted to a different extent in the case of using the above-mentioned culture media 1# to 7# than in the case of using the basal medium. The proliferation effect is remarkably improved when the kidney cancer primary cells are cultured by using a kidney cancer primary cell culture medium containing insulin, Y-27632, fibroblast growth factor (FGF-10), fibroblast growth factor 9 (FGF-9), non-essential amino acids, B27 and TrypLE Express additives.
Different concentration experiments prove that the proliferation effect is optimal when the insulin in the culture medium 1# to 7# is 15ug/ml, the fiber growth factor (FGF-10) is 5ng/ml, the fiber growth factor 9 (FGF-9) is 50ng/ml, the nonessential amino acid is 200uM, the B27 is 1:50 and the nicotinamide is 20 uM.
The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions within the technical scope of the present disclosure should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims (7)

1. A kidney cancer primary cell culture medium, characterized in that:
consists of an initial culture medium, a ROCK inhibitor, nicotinamide, antibiotics, insulin, nonessential amino acids, B27, glutamine, fetal bovine serum and specific additive factors;
the initial culture medium is selected from GGT551, DMEM/F12 and RPMI-1640 culture medium;
the ROCK inhibitor is Y-27632, and the concentration range of the Y-27632 is 5-20 uM;
the concentration range of the nicotinamide is 5-40 uM;
the antibiotics comprise penicillin and streptomycin, and the concentration ranges of the penicillin and the streptomycin are 20-200 mug/mL;
the concentration range of the insulin is 2-30 mug/mL;
the total concentration range of the nonessential amino acids is 30-400 mu M;
the volume ratio of the B27 relative to the culture medium is 1:10-1:200;
the concentration range of the glutamine is 0.1-10 mM;
the volume ratio of the fetal bovine serum to the culture medium is 1:5-1:20;
the concentration range of the specific additive factors is 10-100 ng/ml.
2. The primary cell culture medium for renal cancer of claim 1, wherein:
the concentration range of the Y-27632 is 5-10 uM;
the concentration range of the nicotinamide is 10-20 uM;
the concentration ranges of the penicillin and the streptomycin are 50-100 mug/mL;
the concentration range of the insulin is 10-20 mug/mL;
the total concentration range of the nonessential amino acids is 100-300 mu M;
the volume ratio of the B27 relative to the culture medium is 1:20-1:100;
the concentration of the glutamine is 1-5 mM;
the volume ratio of the fetal bovine serum to the culture medium is 1:5-1:10;
the concentration range of the specific additive factors is 30-70 ng/ml.
3. The primary cell culture medium for renal cancer of claim 1 or 2, wherein:
the specific additive factors comprise a fibroblast growth factor (FGF-10) and a fibroblast growth factor 9 (FGF-9), and the volume ratio of the fibroblast growth factor (FGF-10) to the fibroblast growth factor 9 (FGF-9) is 1:10-1:20.
4. A primary cell culture medium for renal cancer according to claim 3, wherein:
the non-essential amino acids include glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine.
5. A method for culturing primary cells of kidney cancer, characterized by:
culturing gastric cancer primary cells using the kidney cancer primary cell culture medium according to any one of claims 1 to 4.
6. The method for culturing primary cells of kidney cancer according to claim 5, wherein:
in the culture, the cell density is 1-4×10 4 Individual/cm 2 The trophoblasts are added.
7. The method for culturing primary cells of kidney cancer according to claim 6, wherein:
the trophoblast is an irradiated NIH-3T3 cell, the irradiation source is X-ray or gamma-ray, and the irradiation dose is 20-30 Gy.
CN202410132897.7A 2024-01-31 2024-01-31 Culture medium and culture method of kidney cancer primary cells Pending CN117757749A (en)

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