CN105154405B - A kind of colorectal cancer cell primary culture method of low damage - Google Patents
A kind of colorectal cancer cell primary culture method of low damage Download PDFInfo
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Abstract
The present invention relates to a kind of colorectal cancer cell primary culture methods of low damage comprising following steps: (1) organizing pre-treatment: by Colorectal Carcinoma through antibiotic treatment；(2) stripping and slicing；(3) it cultivates: the tissue block cut being put into antibiotic culture medium and carries out initial-stage culture and normal culture, until cell is adherent.Method of the invention is small to cellular damage, cell survival rate is high, easy culture easy to operate.
Description
Technical field
The invention belongs to technical field of cell culture, and in particular to a kind of colorectal cancer cell originally culture side of low damage
Method.
Background technique
Colorectal cancer is common alimentary system malignant tumour, and the morbidity and mortality of China's colorectal cancer are in recent years
Rise year by year trend.Most colorectal cancer patients have belonged to middle and advanced stage when medical, treatment cost is big, effect is poor, long term survival rate is low.
Heavy burden is brought to patient, family and society simultaneously.Colorectal cancer have obvious cancer before and cancer early lesion,
And the time was up to for more than ten years.This provides chance for the early detection of colorectal cancer and precancerous lesion and early treatment.Tumour mark
The measurement of will object plays an important role in terms of the diagnosis of tumour, curative effect evaluation, detection recurrence and supposition, to raising
Clinical tumor treatment level has great importance.
Colorectal cancer is polygenes change procedure, and unique identification object screening specificity and sensibility are lower, it is difficult to
Cope with the screening of large sample crowd.Although many for the research in terms of colorectal cancer tumor markers both at home and abroad, at present
Applied to clinical colorectal cancer tumor markers --- CEA, CA199, CA125, CA724 etc. still lack enough sensitivitys and
Specificity finds the new tumor marker closely related with colorectal cancer development process, to early diagnosis and intervenes Colon and rectum
Cancer is of great significance.But so far, it is straight not yet to find that not only sensitive but also special tumor markers can be applied to clinical knot
The detection of intestinal cancer.Therefore, effective tumor markers are found as primary dcreening operation means, progress colorectal cancer early screening, early stage diagnosis and treatment
To block or delay colorectal cancer progress, improve patient's prognosis and extend life cycle be of great significance.
By the high flux screening for expressing Colorectal Carcinoma cell and normal Colon and rectum mucomembranous cell albumen, it is possible to
It was found that the difference of tumor tissues and normal mucosa histocyte protein expression profiles, and then find new tumor markers.It is high-throughput
The albumen of screening tumor cells expression then needs to be related to the originally culture of tumour cell, and this research method is to primary training
The fostering requirement for supporting cell is very high, and Yao Jinliang avoids caused cell rupture or cell membrane damage in experimental implementation.
Originally culture is to be cultivated immediately after directly removing cell, tissue and organ from body, in colorectal cancer
In research, the originally culture of cancer cell has not been reported directly to be grown using mechanical crushing method, is all made of traditional enzyme digestion,
Machinery is carried out to Colorectal Carcinoma early period to shred, enzymic digestion then is added into the tissue shredded, is dispersed into it individually
Cell.
Traditional enzyme digestion has the disadvantage that (sample can only be selected identical due to digestion time
The time is handled, for example bigger tissue block is digested to single cell as standard formulation digestion time, is necessarily caused lesser
The digestion of tissue block and free cell is excessive), it will cause cell dissociation and unevenly even digest excessively, damaging cells film destroys
Cell influences cell activity, or digestion is not thorough the progress for being unfavorable for high flux screening experiment；Enzyme it is many kinds of, disappearing
Select the type of enzyme and concentration that there is variability when change, different its requirement to enzyme of tissue are also different, need the item of complexity
Part is groped.
Summary of the invention
Defect based on the prior art, the technical problem to be solved by the invention is to provide a kind of pair of cellular damage is small, thin
Born of the same parents survival rate is high, the low damage easy to operate easily cultivated colorectal cancer cell primary culture method.
The technical scheme adopted by the invention to solve the technical problem are as follows: a kind of colorectal cancer cell originally culture of low damage
Method comprising following steps:
(1) tissue pre-treatment: by Colorectal Carcinoma through antibiotic treatment；
(2) stripping and slicing；
(3) it cultivates: the tissue block cut being put into antibiotic culture medium and carries out initial-stage culture and normal culture, until
Cell is adherent.
Further, step (1) rinses Colorectal Carcinoma with sterile saline, is then placed in antibiotic nothing
Blood serum medium impregnates.
Further, antibiotic in the antibiotic serum free medium are as follows: 500 U/mL penicillin, 500 μ
G/mL streptomysin, 1.25 μ g/mL amphotericin Bs, 80 μ g/mL gentamicins.
Further, step (2) stripping and slicing are as follows: tissue block is put into Tissue Culture Dish, is cut tissue with aseptic operation knife
It is broken to volume 1-50mm3, preferably 15-30mm3。
Further, step (3) initial-stage culture uses initial-stage culture base, contains: volume fraction is 10% tire ox blood
Clearly；100 U/mL penicillin；100 μ g/mL streptomysins；0.25 μ g/mL amphotericin B；80 μ g/mL gentamicins；
2mmol/L glutamine.
Further, step (3) the normal culture uses normal incubation medium, and contain: volume fraction is 10% tire ox blood
Clearly；100 U/mL penicillin；100 μ g/mL streptomysins；0.25 μ g/mL amphotericin B；2 mmol/L glutamine.
Further, -80 DEG C of precooled Tissue Culture Dish are used when step (2) stripping and slicing；When organizing stripping and slicing, cell is trained
Feeding ware is placed on ice, and tissue block is placed in Tissue Culture Dish, and low-temperature operation can delay cell metabolism procedure, thus relatively long
Ground saves organizational vitality, effectively avoid tissue in vitro after ischemic, cell caused by anoxic.
Further, while the tissue of step (1) is through antibiotic treatment, through interleukin 6 inhibitor immersion treatment.
Specifically include following operating procedure:
(1) tissue pre-treatment: Colorectal Carcinoma is chosen, sterile saline rinses 30s or more, with highly concentrated with 4 kinds
The serum-free RPMI-1640 culture medium for spending antibiotic and interleukin 6 inhibitor impregnates；After 5-10min, replace fresh containing high concentration
5-10min is impregnated again after the serum-free RPMI-1640 culture medium of antibiotic.In soaking process, rock frequently, it is reverse to rock, 6
Beat/min, more preferably to contact with each other and clean；Interleukin 6 inhibitor selects Siltuximab in soak, and concentration is 1-15 μ
G/mL, preferably 11 μ g/mL；
This step operation can neutralize the interleukin 6 in necrotic tissue, reduce the cellular damage of its mediation, while this is operated
Reduce the materials standard of tissue；Necrotic tissue is less than the one third of sample total volume, and conventional method requires to avoid bad
Dead tissue, with the progress of medicine, for the clinical materials for the object that swell from now on just vulnerable to limitation, reducing materials standard to be undoubtedly is scientific research
Work provides premise and guarantee, substantially increases originally culture success rate；
4 kinds of antibiotic concentrations in step (1):
500 U/mL penicillin,
500 μ g/mL streptomysins,
1.25 μ g/mL amphotericin Bs,
80 μ g/mL gentamicins；
(2) stripping and slicing: tissue block is put into -80 DEG C of precooled Tissue Culture Dish, when organizing stripping and slicing, by Tissue Culture Dish
It is placed on ice, tissue block is placed in Tissue Culture Dish, with aseptic operation knife minced tissue to volume 1-50mm3, preferably 15-
30mm3；
(3) without culture medium culture: pure blood is clear and rich to wash culture bottle, and tissue block is placed directly into culture bottle, and tissue cultures rise
The 3h of beginning does not add culture medium, in order to accelerate adherent and adherent stronger, after 3h, adhere to culture to tissue block
It on bottle, then adds 1mL pure blood and is cultivated clearly, pure serum free culture system is for 24 hours；
(4) initial-stage culture: being 10% fetal calf serum, 4 kinds of Antibiotics of Low Concentration and 2 mmol/L paddy ammonia with containing volume fraction
The RPMI-1640 culture medium of amide continues to cultivate 48-72h, wherein 4 kinds of Antibiotics of Low Concentration:
100 U/mL penicillin,
100 μ g/mL streptomysins,
0.25 μ g/mL amphotericin B,
80 μ g/mL gentamicins；
(5) normal culture: being 10% fetal calf serum, 3 kinds of antibiotic and 2 mmol/L glutamine with containing volume fraction
RPMI-1640 culture medium changes liquid Nostoc commune Vanch, changes liquid within every 5-7 days；Observe the adherent situation of cell；3 kinds of Antibiotics of Low Concentration:
100 U/mL penicillin,
100 μ g/mL streptomysins,
0.25 μ g/mL amphotericin B；
(6) after confirmation cell is adherent, primary part observation culture changes liquid according to concrete condition.
RPMI-1640 culture medium of the invention is this field common culture medium, and for details, reference can be made to " E16.5 mice embryonic pancreases
Glandular tissue transplantation treatment artificial diabetes ", Chinese of Journal General Surgery, in September, 2013, the 9th phase of volume 28.
The beneficial effects of the present invention are:
(1) it when materials, does not need deliberately to avoid necrotic tissue, improves materials efficiency, avoid waste of material；
(2) tissue block is put into Tissue Culture Dish, with aseptic operation knife minced tissue, reduces the machinery to tissue block
Damage, in addition, the tissue block size requirements to chopping are not stringent, conventional method requires chopping to 1 mm3Hereinafter, this method volume
Biggish tissue block can also reach culture effect, and 15-30mm3Tissue block effect ratio 1mm3More preferably；
(3) according to the special generation environment of Colorectal Carcinoma, specific antibiotic concentration is configured, and according to cancer cell
Growth cycle characteristic changing concentration and type, be conducive to inhibit tissue in bacterial reproduction, avoid cell microbiological contamination, improve patch
Wall rate；
(4) serum rinse culture bottle and 3h no liquid culture promote quick wall attaching；First day quickening tissue of pure serum free culture system
Adaptation to vitro culture conditions；
(5) glutamine guarantees fissional raw material supply.
The present invention carries out originally culture to Colorectal Carcinoma using mechanical stripping and slicing, the specific culture solution of cooperation, avoids
The high risk of tradition machinery breaking method contamination rate also avoids damage of the enzyme digestion to cell, ensure that greatest extent
The integrality of cell is realized and is quickly repaired.Inventor is also found surprisingly that in test the culture incipient stage carries out 3h no liquid
Culture, can accelerate the speed of tissue block adherent, and make adherent stronger, and tissue block is not easily to fall off, can accelerated cell growth.
Acute inflammation and necrotic tissue situation are complicated in Colorectal Carcinoma, and the addition of interleukin 6 inhibitor is deposited when can permit materials
In necrotic tissue.
Detailed description of the invention
Fig. 1 is 1 microscopically observation result of embodiment；
Fig. 2 is comparative example microscopically observation result.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, implement below in conjunction with the present invention
Specific embodiment in example, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described
Embodiment be only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ability
Domain those of ordinary skill every other embodiment obtained without making creative work, belongs to guarantor of the present invention
The range of shield.
Embodiment 1
A kind of colorectal cancer cell primary culture method of low damage of embodiment 1 comprising following steps:
(1) tissue pre-treatment: Colorectal Carcinoma is chosen, sterile saline rinses 30s or more, with highly concentrated with 4 kinds
The serum-free RPMI-1640 culture medium for spending antibiotic and interleukin 6 inhibitor impregnates；After 5-10min, replace fresh containing high concentration
5-10min is impregnated again after the serum-free RPMI-1640 culture medium of antibiotic；In soaking process, rock frequently, it is reverse to rock, 6
Beat/min, more preferably to contact with each other and clean；Interleukin 6 inhibitor selects Siltuximab in soak, and interleukin 6 inhibits
Johson & Johnson's production is selected in agent, and concentration is 11 μ g/mL；
This step operation can neutralize the interleukin 6 in necrotic tissue, reduce the cellular damage of its mediation, while this is operated
Reduce the materials standard of tissue；Necrotic tissue is less than the one third of sample total volume, and conventional method requires to avoid bad
Dead tissue, with the progress of medicine, for the clinical materials for the object that swell from now on just vulnerable to limitation, reducing materials standard to be undoubtedly is scientific research
Work provides premise and guarantee, substantially increases originally culture success rate；
4 kinds of antibiotic concentrations:
500 U/mL penicillin,
500 μ g/mL streptomysins,
1.25 μ g/mL amphotericin Bs,
80 μ g/mL gentamicins.
(2) stripping and slicing: tissue block is put into -80 DEG C of precooled Tissue Culture Dish, when organizing stripping and slicing, by Tissue Culture Dish
It is placed on ice, tissue block is placed in Tissue Culture Dish, with aseptic operation knife minced tissue to volume 15mm3。
(3) without culture medium culture: pure blood is clear and rich to wash culture bottle, and tissue block is placed directly into culture bottle, and tissue cultures rise
The 3h of beginning does not add culture medium, in order to accelerate adherent and adherent stronger, after 3h, adhere to culture to tissue block
It on bottle, then adds 1mL pure blood and is cultivated clearly, pure serum free culture system is for 24 hours.
(4) initial-stage culture: being 10% fetal calf serum, 4 kinds of Antibiotics of Low Concentration and 2 mmol/L paddy with containing volume fraction
The RPMI-1640 culture medium of glutamine continues to cultivate 48-72h, wherein 4 kinds of Antibiotics of Low Concentration:
100 U/mL penicillin,
100 μ g/mL streptomysins,
0.25 μ g/mL amphotericin B,
80 μ g/mL gentamicins.
(5) normal culture: being 10% fetal calf serum, 3 kinds of antibiotic and 2 mmol/L glutamine with containing volume fraction
RPMI-1640 culture medium changes liquid Nostoc commune Vanch, changes liquid within every 5-7 days；Observe the adherent situation of cell；3 kinds of Antibiotics of Low Concentration:
100 U/mL penicillin,
100 μ g/mL streptomysins,
0.25 μ g/mL amphotericin B.
(6) after confirmation cell is adherent, primary part observation culture changes liquid according to concrete condition.
RPMI-1640 culture medium of the invention is this field common culture medium, and for details, reference can be made to " E16.5 mice embryonic pancreases
Glandular tissue transplantation treatment artificial diabetes ", Chinese of Journal General Surgery, in September, 2013, the 9th phase of volume 28.
Embodiment 2
When 2 difference from Example 1 of embodiment is step (1) tissue pre-treatment, interleukin 6 inhibitor
The concentration of Siltuximab is 1 μ g/mL；Step (2) stripping and slicing is to volume 50mm3。
Embodiment 3
When 3 difference from Example 1 of embodiment is step (1) tissue pre-treatment, interleukin 6 inhibitor
The concentration of Siltuximab is 15 μ g/mL；Step (2) stripping and slicing is to volume 1mm3。
Embodiment 4
4 difference from Example 1 of embodiment is step (2) stripping and slicing to volume 30mm3。
Comparative example
The primitive cell culture of Colorectal Carcinoma is carried out using enzyme digestion, concrete operations are as follows:
1、1-2cm3Tissue block is put into Tissue Culture Dish, is added 10mL RPMI-1640 culture medium (serum-free), with height
Pressure sterilizing operating scissors removal nonneoplastic tissue and necrotic tissue；
2, tissue is transferred in new culture dish, is cut into small pieces.20mL RPMI-1640 culture medium (serum-free) resuspension group
It knits block and is transferred in 50mL conical pipe.Room temperature, 1200rpm 6min abandon supernatant；
3, tissue, 37 DEG C of digestion 4hr, with stirring are resuspended in 10mL Tumor Cell Digestion Solution；
4,10mL Tumor Cell Suspension Solution is added and puts out mixing.The filtering of 100mm filter is mixed
Suspension collects filtrate into a clean 50mL conical pipe；
5, room temperature, 1200rpm 8min abandon supernatant.It is resuspended with 20mL Tumor Cell Suspension Solution
Precipitating mixes, obtains the cell mixture of mean value；
6, it is added in the new 50mL conical pipe of 20mL Tumor Cell Purification Solution to one, is labeled as
Pipe A, room temperature 1200rpm 2min；
7, gained 20mL cell suspension in 5 is placed in the solution upper layer of pipe A；
8, pipe A is vertically stood into 6min in room temperature；
9, pipetting tip is carefully protruded into conical pipe bottom, siphons away 6mL Tumor Cell Purification from bottom
Solution moves in a new 50mL conical pipe, is designated as pipe B；
10,1200rpm 8min abandons supernatant；
11, (volume fraction is 10% fetal calf serum, 100 U/mL penicillin, 100 μ g/ to 5mL RPMI-1640 culture medium
ML streptomysin) precipitating is resuspended；
12, cell is moved to a hole of 6 orifice plates, is cultivated 3-4 days.
Effect example
By same Colorectal Carcinoma, the method that embodiment 1 and comparative example is respectively adopted carries out originally culture, comparative example warp
Step 12 processing, culture have no cell Proliferation for 3-4 days, are compared after normal culture 20 days after culture 20 days with embodiment 1,
As a result as shown in Figure 1, Figure 2 and table 1.
The cultivation results of table 1 embodiment 1 and comparative example
By comparison, it was found that:
Kit (enzyme digestion) is more demanding to tissue block, and complex for operation step, operation takes a long time, in vitro from tissue
To starting to cultivate, 6-7h is needed, tumor tissues are detached from the environment of suitable growth for a long time.In terms of final result, when enzymic digestion pair
The damage of tissue is very big, and final gained cell quantity seldom (less than the 5/visual field), far can not reach the number of cell culture
Amount, and the structure of gained cell has obvious damage, is not able to satisfy experiment demand.
Method of the invention only need to tentatively shred tissue block, and not stringent to the size requirements of tissue block,
Reduce shearing number, alleviate the mechanical damage degree to tissue block, then disappears without carrying out enzyme to the tissue block after shearing
Change, further reduces the damage to cell.After preliminary immersion shearing, it can be cultivated, be cultivated in vitro since tissue,
0.5h is not exceeded, reduces tumor tissue cell to greatest extent in the time of non-suitable growth environment.In terms of final result,
After the processing of equiponderant tissue block, cell quantity is more than traditional enzyme digestion (more than the 70/visual field) under same field of view, can be with
Meet experiment demand in next step.
Claims (4)
1. a kind of colorectal cancer cell primary culture method of low damage, which is characterized in that it includes the following steps:
(1) tissue pre-treatment: by Colorectal Carcinoma through antibiotic treatment, while through interleukin 6 inhibitor immersion treatment；
(2) stripping and slicing；
(3) it cultivates: first carrying out no culture medium culture, pure blood is clear and rich to wash culture bottle, and tissue block is placed directly into culture bottle, tissue
The 3h for cultivating starting, does not add culture medium, after 3h, adheres on culture bottle to tissue block, then add 1mL pure blood and trained clearly
It supports, pure serum free culture system is for 24 hours；It is then placed in antibiotic culture medium and carries out initial-stage culture and normal culture, until cell is adherent；
Step (3) the primary culture uses primary culture medium, and it is 10% fetal calf serum, 4 that primary culture medium, which is containing volume fraction,
The RPMI-1640 culture medium of kind Antibiotics of Low Concentration and 2mmol/L glutamine, wherein 4 kinds of Antibiotics of Low Concentration are 100 U/
ML penicillin, 100 μ g/mL streptomysins, 0.25 μ g/mL amphotericin B, 80 μ g/mL gentamicins；
Step (3) the normal culture uses normal incubation medium, and it is 10% fetal calf serum, 3 that normal incubation medium, which is containing volume fraction,
The RPMI-1640 culture medium of kind of antibiotic and 2 mmol/L glutamine, wherein 3 kinds of antibiotic be 100 U/mL penicillin,
100 μ g/mL streptomysins, 0.25 μ g/mL amphotericin B.
2. the colorectal cancer cell primary culture method of low damage according to claim 1, which is characterized in that step (1)
Colorectal Carcinoma is rinsed with sterile saline, antibiotic serum free medium is then placed in and impregnates.
3. the colorectal cancer cell primary culture method of low damage according to claim 2, which is characterized in that described containing anti-
Antibiotic in the serum free medium of raw element are as follows: 500 U/mL penicillin, 500 μ g/mL streptomysins, 1.25 μ g/mL both sexes
Mycin B, 80 μ g/mL gentamicins.
4. the colorectal cancer cell primary culture method of low damage according to claim 1, which is characterized in that step (2)
Stripping and slicing are as follows: tissue block is put into Tissue Culture Dish, is shredded tissue to volume 15-30mm with aseptic operation knife3。
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| CN108396010A (en) * | 2017-02-06 | 2018-08-14 | 王琼 | A kind of extracorporeal culturing method of colorectal cancer organoid |
| CN108103023A (en) * | 2018-02-01 | 2018-06-01 | 河北医科大学第医院 | People's carcinoma of the rectum primary cell and its application |
| CN108192871A (en) * | 2018-02-01 | 2018-06-22 | 河北医科大学第医院 | People's carcinoma of the rectum primary cell and its application |
| CN108192872A (en) * | 2018-02-01 | 2018-06-22 | 河北医科大学第医院 | People's carcinoma of the rectum primary cell and its application |
| CN108103024A (en) * | 2018-02-01 | 2018-06-01 | 河北医科大学第医院 | People's carcinoma of the rectum primary cell and its application |
| CN110592018A (en) * | 2018-06-13 | 2019-12-20 | 北京吉尚立德生物科技有限公司 | Method for culturing primary cells of colorectal cancer solid tumors |
| CN110029089A (en) * | 2019-04-29 | 2019-07-19 | 北京和合医学诊断技术股份有限公司 | Serum free medium, preparation method and the method for cultivating primary tumor cell |
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