CN108486059A - A kind of method for building up of cell culture fluid and the primary lung cancer cell line of people - Google Patents
A kind of method for building up of cell culture fluid and the primary lung cancer cell line of people Download PDFInfo
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- CN108486059A CN108486059A CN201810408025.3A CN201810408025A CN108486059A CN 108486059 A CN108486059 A CN 108486059A CN 201810408025 A CN201810408025 A CN 201810408025A CN 108486059 A CN108486059 A CN 108486059A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/40—Nucleotides, nucleosides, bases
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/33—Insulin
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/40—Regulators of development
- C12N2501/405—Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
Abstract
The invention discloses a kind of cell culture fluids, it is characterised in that addition fetal calf serum, kinase inhibitor Y 27632, Gl μ taMAX, insulin, hydrocortisone, epidermal growth factor and adenine in 12 culture mediums of F and DMEM culture medium mixtures.Tissue block inner cell can be promoted adherent by the use of the F culture solutions of offer and grown, but can also promote fibroblastic growth simultaneously；The cell culture processes energy high success rate of use establishes the primary lung cancer tumor cell of people, and culture is time-consuming short, and cell can be passed on repeatedly, be expanded.Important material source is provided for the basic research and relevant translational medicine application of lung cancer.With very important realistic meaning and practical value.
Description
Technical field
The present invention relates to the foundation of biotechnology more particularly to a kind of cell culture fluid and the primary lung cancer cell line of people
Method.
Background technology
At present in tumor cell culture technology, to tumour cell carry out original cuiture and build be most common technology be application
The basal medium of the fetal calf serum (can abbreviation FCS) of addition 10%, such as RPMI-1640 cultivate tumour cell.But
Apply the tumor cell culture technology culture primary tumor cell, success rate very low at present.For example, using this medium culture
Primary lung cancer and prostate gland cancer cell, success rate are below 10%, and it is lower to pass on the success rate built and be.Therefore, swollen at present
The cell line that mostly application has been built up in tumor research.Since built cell line have passed through long-term in vitro culture and biography more
In generation, they have lost the original biological characteristics of many tumour cells, and this greatly limits the biological characteristics of tumour to grind
Study carefully.In addition, nowadays immunotherapy of tumors progress is rapid, autologous tumor cell is identification, separation, obtains patient tumors mutant antigen
Important materials；It is also detection patient gene's mutation, realizes the basis precisely treated.Therefore more efficiently Primary Tumor is established
Cell culture processes contribute to the development and application of multinomial the relevant technologies.
Some primary tumor cell culture solutions of latest developments make the success rate of a variety of primitive cell cultures increase.But
And a kind of general, for cancerous lung tissue sample cell culture processes are not set up.And for main mixed in incubation
Miscellaneous ingredient, fibroblastic removal is also without simple, effective scheme.
Invention content
For disadvantages described above, present invention aims at how to improve primary tumor cell culture solution a variety of primary cells is made to train
Foster success rate.
To achieve the goals above, the present invention provides a kind of cell culture fluids, it is characterised in that F-12 culture mediums with
Fetal calf serum and kinase inhibitor Y-27632 are added in DMEM culture medium mixtures.
The cell culture fluid, it is characterised in that further include Gl μ taMAX, insulin, hydrocortisone, epidermal growth
The factor and adenine.
The cell culture fluid, it is characterised in that F-12 culture mediums press volume proportion 3 with DMEM culture mediums：1 is mixed
Symphysis at mixed culture medium, then add the fetal calf serum of volumetric concentration 3%~10%, 2mM Gl μ taMAX, 5 μ g/mL insulin,
0.4 μ g/mL hydrocortisones, 10ng/mL epidermal growth factor, 24 μ g/mL adenines and 2.5~10 μm of ol/L Rho kinases suppressions
Preparation Y-27632.
A kind of method for building up of the primary lung cancer cell line of people, it is characterised in that will be from the sampled sample group of patients with lung cancer
It knits fragment and turns to Tumor fragments, Tumor fragments are washed using 1640 culture medium, the Tumor fragments after washing are resuspended in
Tumor fragments suspension is formed in cell culture fluid, and Tumor fragments suspension is inoculated into be placed in cell incubator after orifice plate and is cultivated
2~5 days, reject culture solution, non-attached tumor fragment and suspension cell, using the cell culture fluid pair containing 50% μ g/mL G418
Tumor fragments carry out culture in 5~10 days, replace within every 2~3 days the cell culture fluid for once containing 50 μ g/mL G418, cultivate to every
Hole cell convergence degree is not less than 90%.
The method for building up of the primary lung cancer cell line of the people, it is characterised in that increase removal fiber finer before amplification cultivation
Born of the same parents operate, and fibroblast are removed specifically by trypsin digestion, specially after reject cell culture fluid, using without blood
Clear DMEM culture mediums wash cell, after discarding cleaning solution, add 0.25% trypsin-EDTA solutions warmed in advance, will
Cell is placed in cell incubator, stands 2 minutes, and the DMEM culture mediums containing 10% fetal calf serum are added and terminate enzyme reaction, with shifting
Liquid rifle gently purges attached cell, and fibroblast is made to be detached from tissue culture plate；Abandon supernatant, then with containing 10% fetal calf serum
DMEM culture mediums washing cell is primary, abandons supernatant；Again it is swollen to continue culture for cell culture fluid of the addition containing 50 μ g/mL G418
Oncocyte.
The method for building up of the primary lung cancer cell line of the people, it is characterised in that using 1640 culture medium to Tumor fragments into
Row washing be specially will be fragmented after model tissue be resuspended in be placed in centrifuge tube in 1640 culture medium and wash.
The method for building up of the primary lung cancer cell line of the people, it is characterised in that the cell culture fluid in each hole of orifice plate
Amount is 300~700 μ L.
The use of F culture solutions provided by the invention can promote tissue block inner cell adherent and grow, but can also promote simultaneously
Fibroblastic growth；The cell culture processes energy high success rate of use establishes the primary lung cancer tumor cell of people, culture consumption
When it is short, cell can be passed on repeatedly, be expanded.Important material is provided for the basic research of lung cancer and relevant translational medicine application
Source.With very important realistic meaning and practical value.
Description of the drawings
Fig. 1 is the method flow diagram for cultivating the primary lung carcinoma cell of people；
Fig. 2 is the light microscopic figure of the primary lung carcinoma cell of the culture of the 4 different patients obtained.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other without creative efforts
Embodiment shall fall within the protection scope of the present invention.
In order to cultivate the primary lung carcinoma cell of people, specific cell culture fluid is needed to configure, abbreviation F culture solutions are former for people
For the culture of lung carcinoma cell.The culture solution includes F-12 culture mediums and DMEM culture mediums by (3:1, v/v) ratio mixing mixes
Culture medium is closed, the fetal calf serum of 5% (v/v) of addition in the mixed culture medium, 2mM Gl μ taMAX, 5 μ g/mL insulin, 0.4
μ g/mL hydrocortisones, 10ng/mL epidermal growth factor, 24 μ g/mL adenines and 5 μm of ol/L Rho kinase inhibitors (Y-
27632).The use of F culture solutions can promote tissue block inner cell adherent and grow, but can also promote fibroblastic life simultaneously
It is long.
Fig. 1 is the method flow diagram for cultivating the primary lung carcinoma cell of people：
Cancerous lung tissue is cut into fragment by step 1.Operating scissors can be used by taking the tissue of size about 1cm patients with lung cancer
Cut into tiny Tumor fragments.
Step 2, washing Tumor fragments to supernatant are clarified.Cancerous lung tissue fragment is resuspended in 1640 culture medium and is placed in
In 15mL centrifuge tubes.After Tumor fragments natural subsidence, muddy supernatant liquid is siphoned away, tumour is washed repeatedly with 1640 culture medium
Fragment is clarified to supernatant liquid twice or repeatedly.
Tumor fragments are resuspended in F culture solutions, and are inoculated in 6 orifice plates by step 3, and each hole is inoculated with 500 μ L.Such as it will be from
The Tumor fragments of heart bottom of the tube are resuspended in 6mL (I) described culture solution, are seeded in 2 pieces of 6 orifice plates.500 μ L tumours are inoculated with per hole
Fragment suspension.
Step 4, basis culture, the orifice plate of inoculated tumour fragment are placed in cell incubator after cultivating 5 days, discard F trainings
Nutrient solution simultaneously removes extra non-attached tumor fragment and suspension cell.Per hole, addition 1mL contains the F culture solutions of 50 μ g/mL G418,
Culture 5-10 days in cell incubator are continued at, F culture solution of the replacement containing 50 μ g/mL G418 1 time per 2-3 days.To every hole cell
When convergence degree reaches 90%.
Step 5, removal fibrocyte operation.It is frozen using each hole cell of trypsin digestion or is passaged to after being merged
F culture solutions of the 1mL containing 50 μ g/mL G418 is used to continue amplification cultivation acquisition lung cancer in the culture dish of 10cm again primary thin
Born of the same parents.
Before primary tumor cell amplification cultivation, increases the operation of removal fibrocyte, can further pass through trypsin digestion
Remove fibroblast.Cell culture fluid is specially discarded, the DMEM culture mediums washing cell without serum is used in combination.Discard washing
After liquid, 0.25% trypsin-EDTA solutions warmed in advance are added, cell is placed in cell incubator, stands 2 minutes,
The DMEM culture mediums containing 10% fetal calf serum are added and terminate enzyme reaction, gently purges attached cell with liquid-transfering gun, makes into fiber finer
Born of the same parents are detached from tissue culture plate.It abandons supernatant, then with the DMEM culture mediums containing 10% fetal calf serum to wash cell primary, abandons supernatant.
It can be removed through washing a large amount of fibroblasts twice, and primary tumor cell is still within adhered state.Again addition contains 50 μ
(I) described culture solution of g/mL G418 continues to cultivate tumour cell.
It is cultivated using F pairs of 16 cancerous lung tissue samples of above-mentioned cultural method and culture solution, is successfully established primary lung cancer
11, cell, to the lung cancer sample of size about 0.5cm, culture success ratio is more than 80%.
Fig. 2 is the light microscopic figure of the primary lung carcinoma cell of the culture of the 4 different patients obtained, shows patient A, B, C respectively
The primary lung carcinoma cell of the culture of patients different with D4.Experiment is proved through remarkable primary lung carcinoma cell cultural method subculture
The cellular morphology of acquisition is good, clear-cut.
The primary lung carcinoma cell cultural method of people has the advantages that compared with prior art:The present invention's is thin
Born of the same parents' cultural method energy high success rate establishes the primary lung cancer tumor cell of people, and culture is time-consuming short, and cell can be passed on repeatedly, be expanded.
Important material source is provided for the basic research and relevant translational medicine application of lung cancer.With very important realistic meaning
And practical value.For example, using cell culture processes of the present invention, it is 5 days most short in i.e. visible lung cancer cell grow.
And can effectively inhibit or remove the major impurity cell in primitive cell culture by adding G418 and trypsin treatment, at fibre
Cell is tieed up, and the processing does not influence the growth of primary tumor cell.
Above disclosed is only an embodiment of the present invention, cannot limit the right model of the present invention with this certainly
It encloses, those skilled in the art can understand all or part of the processes for realizing the above embodiment, and is wanted according to right of the present invention
Equivalent variations made by asking still fall within the range that the present invention is covered.
Claims (7)
1. a kind of cell culture fluid, it is characterised in that in F-12 culture mediums and DMEM culture medium mixtures addition fetal calf serum and
Kinase inhibitor Y-27632.
2. cell culture fluid according to claim 1, it is characterised in that further include Gl μ taMAX, insulin, hydrogenation can
Pine, epidermal growth factor and adenine.
3. cell culture fluid according to claim 2, it is characterised in that F-12 culture mediums are matched with DMEM culture mediums by volume
Than 3：1, which carries out mixing, generates mixed culture medium, then adds the fetal calf serum of volumetric concentration 3%~10%, 2mM Gl μ taMAX, 5 μ
G/mL insulin, 0.4 μ g/mL hydrocortisones, 10ng/mL epidermal growth factor, 24 μ g/mL adenines and 2.5~10 μm of ol/
L Rho kinase inhibitors Y-27632.
4. a kind of method for building up of the primary lung cancer cell line of people, it is characterised in that will be from the sampled sample tissue of patients with lung cancer
Fragment turns to Tumor fragments, is washed to Tumor fragments using 1640 culture medium, the Tumor fragments after washing are resuspended in carefully
Tumor fragments suspension is formed in born of the same parents' culture solution, the cell culture fluid is the cell culture described in claims 1 to 3 any one
Tumor fragments suspension is inoculated into after orifice plate to be placed in cell incubator and cultivate 2~5 days by liquid, reject culture solution, not adherent swollen
Tumor fragment and suspension cell, the culture using the cell culture fluid containing 50 μ g/mLG418 to Tumor fragments progress 5~10 days, every 2
The cell culture fluid for once containing 50 μ g/mL G418 is replaced within~3 days, culture is not less than 90% to per hole cell convergence degree.
5. the method for building up of the primary lung cancer cell line of people according to claim 4, it is characterised in that increase before amplification cultivation
Add removal fibrocyte operation, fibroblast, specially reject cell culture fluid are removed specifically by trypsin digestion
Afterwards, cell is washed using the DMEM culture mediums without serum, after discarding cleaning solution, adds 0.25% tryptose warmed in advance
Enzyme-EDTA solution, cell is placed in cell incubator, stands 2 minutes, and it is whole that the DMEM culture mediums containing 10% fetal calf serum are added
Only enzyme reaction gently purges attached cell with liquid-transfering gun, and fibroblast is made to be detached from tissue culture plate；Abandon supernatant, then with containing
The DMEM culture mediums washing cell of 10% fetal calf serum is primary, abandons supernatant；Again cell of the addition containing 50 μ g/mL G418 is trained
Nutrient solution continues to cultivate tumour cell.
6. the method for building up of the primary lung cancer cell line of people according to claim 4 or 5, it is characterised in that using 1640 cultures
Liquid to Tumor fragments washed specially will be fragmented after model tissue be resuspended in 1640 culture medium and be placed in centrifuge tube
Inside washed.
7. the method for building up of the primary lung cancer cell line of people according to claim 6, it is characterised in that each hole of orifice plate
The amount of cell culture fluid is 300~700 μ L.
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| CN110592022A (en) * | 2019-09-17 | 2019-12-20 | 罗国安 | Special culture medium for lung tumor organoid and 3D culture method without stent |
| CN110680556A (en) * | 2019-11-12 | 2020-01-14 | 鼠来宝(武汉)生物科技有限公司 | Mouse in-vitro fertilization reagent, preparation method thereof and in-vitro fertilization method |
| CN110699324A (en) * | 2019-11-25 | 2020-01-17 | 四川大学华西第二医院 | Mouse fibroblast tumor cell strain and application thereof |
| CN111944737A (en) * | 2019-05-16 | 2020-11-17 | 苏州吉美瑞生医学科技有限公司 | Clinical-grade autologous bronchial basal layer cells, reinfusion preparation and preparation process |
| CN113528445A (en) * | 2021-06-21 | 2021-10-22 | 创模生物科技(北京)有限公司 | PDX modeling adjuvant and application thereof |
| WO2023039999A1 (en) * | 2021-09-15 | 2023-03-23 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium and culture method for lung cancer epithelial cells, and application thereof |
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| CN111944737A (en) * | 2019-05-16 | 2020-11-17 | 苏州吉美瑞生医学科技有限公司 | Clinical-grade autologous bronchial basal layer cells, reinfusion preparation and preparation process |
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| CN111944737B (en) * | 2019-05-16 | 2023-12-26 | 苏州吉美瑞生医学科技有限公司 | Clinical grade autologous bronchial basal layer cell and reinfusion preparation and preparation process |
| CN114196613B (en) * | 2019-05-16 | 2024-03-12 | 苏州吉美瑞生医学科技有限公司 | Application of bronchial basal layer cells in preparation of medicaments for treating bronchiectasis |
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| CN110592022B (en) * | 2019-09-17 | 2021-07-20 | 浙江弘瑞医疗科技有限公司 | Special culture medium for lung tumor organoid and 3D culture method without stent |
| CN110680556A (en) * | 2019-11-12 | 2020-01-14 | 鼠来宝(武汉)生物科技有限公司 | Mouse in-vitro fertilization reagent, preparation method thereof and in-vitro fertilization method |
| CN110699324A (en) * | 2019-11-25 | 2020-01-17 | 四川大学华西第二医院 | Mouse fibroblast tumor cell strain and application thereof |
| CN113528445A (en) * | 2021-06-21 | 2021-10-22 | 创模生物科技(北京)有限公司 | PDX modeling adjuvant and application thereof |
| WO2023039999A1 (en) * | 2021-09-15 | 2023-03-23 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium and culture method for lung cancer epithelial cells, and application thereof |
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