CN101245337A - Primary culture method for maintaining near term growth of large intestinal cancer tumour cell - Google Patents
Primary culture method for maintaining near term growth of large intestinal cancer tumour cell Download PDFInfo
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- CN101245337A CN101245337A CNA2008100597587A CN200810059758A CN101245337A CN 101245337 A CN101245337 A CN 101245337A CN A2008100597587 A CNA2008100597587 A CN A2008100597587A CN 200810059758 A CN200810059758 A CN 200810059758A CN 101245337 A CN101245337 A CN 101245337A
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Abstract
The invention pertains to the primary culture technical field of colorectal cancer tumor cells. The method includes: 1) obtaining a fresh tumor sample by operation, cleaning and cutting the pollution and necrosis areas, immersing in iodophor for disinfection, adopting a PBS cleaning liquid containing dual-antigen for cleaning; 2) cutting the tumor sample into small tissue blocks, still placing, removing the supernatant liquid, using the PBS cleaning liquid containing dual-antigen for cleaning, carrying out centrifugal separation and removing the supernatant liquid; 3) adopting collagenase digestion method or tissue block adhering wall method to carry out the tissue adhering wall and cell climbing out in a culture liquid; 4) continually culturing and carrying out purification processing in the culture liquid to maintain the necessary number for the cell growth. The success rate of the conventional primary culture method for culturing the colorectal cancer tumor cells with good state is very low (6 percent), however, the primary culture method of the invention can achieve more than 33 percent, thus significantly improving the success probability of short-term in vitro culture of the primary colorectal cancer cells; furthermore, the technique is simple and easy, the cost is low, so as to provide a good optional platform for applying the experimental studies of the short-term primary colorectal cancer cells.
Description
Technical field
The invention belongs to the former foster technical field of being commissioned to train of large bowel cancer tumour cell.
Background technology
Up to now, both at home and abroad from Colorectal Carcinoma, separate, purifying tumour cell and the large bowel cancer tumour cell of setting up stable growth all do not obtain high-efficiency method.Though the screening by a large amount of samples finally can be set up clone, huge workload and very long screening time make its culture efficiency quite low.Because in former generation,, tumour can keep the oncobiology characteristic of virgin state well, all significant for tumour research itself and the research of various antitumor drug, it is very urgent to seek a kind of easy and former efficiently breeding method of being commissioned to train, and the research that much is based upon the colorectal carcinoma cell line level also need further be verified its reliability in the primary cell level.Great mass of data shows that the difficult point of former generation large bowel cancer cultivation is: bacterium and inoblast pollute, just for cell growth difficulty, subculture poor growth or stagnation.And maximum difficult point is to influence the subculture cessation of growth cessation of cell long-period growth.Because of a lot of experiments need not the tumor growth of permanent generation, so the cultivation and the purifying of the large bowel cancer primary cell of short-term survival mainly are devoted in this research, can for former generation tumour albumen, DNA, RNA extract research such as partial immunity detection.
Summary of the invention
The object of the invention is to overcome some problems that exist in the prior art, and a kind of technical scheme of keeping the former breeding method of being commissioned to train of large bowel cancer tumour cell term growth is provided.
A kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth is characterized in that comprising following processing step:
1) the fresh state tumor specimen is obtained in operation, and after physiological saline cleaned and cuts off filth and necrotic zone, Iodophor soaking disinfection 5 minutes adopted to contain two anti-PBS scavenging solutions and clean;
2) in containing two anti-PBS scavenging solutions above-mentioned tumor specimen is cut into the fine tissue piece, leaves standstill, go supernatant liquor, clean with containing two anti-PBS scavenging solutions more afterwards, supernatant liquor is removed in centrifugation;
3) employing collagenase digestion or tissue block adherent method are organized adherent in nutrient solution and cell climbs out of, and described nutrient solution is a 10-20% foetal calf serum nutrient solution;
4) in above-mentioned nutrient solution, continue to cultivate and purification process, keep cell and grow to desired number.
Described a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth is characterized in that described collagenase digestion processing step is:
1) according to specimen block amount, by 1: the volume ratio of 8-12 adds collagenase digesting liquid, places 37 ℃ of water-baths to shake case effect 0.5-3 hour, all is half batting shape or fine particle shape to tissue block, leaves standstill, and supernatant liquor is abandoned in the dropper suction;
2) centrifugation is abandoned supernatant liquor with the dropper suction, uses resuspended, the purge mixing of serum-free DMEM nutrient solution then, and is centrifugal, abandons supernatant liquor;
3) in 10-20% foetal calf serum nutrient solution, after 48 hours, discard original fluid, change to the new nutrient solution of capacity, and changed one time nutrient solution in per 3 days with thin quantity of fluid training method cultivation;
Described a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth is characterized in that described tissue block adherent method processing step is:
1) sample tissue after treatment adds resuspended, the cleaning of serum-free DMEM nutrient solution, and supernatant liquor is abandoned in centrifugation;
2) according to specimen block's amount, by 1: the volume ratio of 8-12 adds collagenase digesting liquid, places 37 ℃ of water-baths to shake the case effect 20-40 minute, make matter separation between the tissue block surface, expose tumour cell, clean with serum-free DEME nutrient solution afterwards, staticly settle, abandon supernatant liquor;
3) add pure foetal calf serum (FBS), mixing fully soaks into the sample tissue;
4) plantation of tissue block: the tissue block that pure foetal calf serum (FBS) is soaked is drawn in culturing bottle or the culture dish, be evenly distributed, continue to drip pure foetal calf serum (FBS) to covering culturing bottle or culture dish bottom surface fully, culturing bottle or the slow heeling condition of culture dish are placed in the incubator, and it is adherent that tissue block is sticked;
5) after 6-12 hour, add 10-20% foetal calf serum nutrient solution, tissue block just is submerged and is unlikely to float, the level position is put into incubator and is cultivated again, how much changes one time nutrient solution every 24-48 hour according to tissue block.To tissue block adherent firmly after, add the capacity nutrient solution and changed liquid once in per 72 hours.
Described a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth is characterized in that described purification process adopts the mechanical curettage method, and its processing step is:
1) mark: microscopically is observed, with the position of marker pen growth tumour cell under the circle of the back side of culturing bottle or culture dish;
2) strike off: discard nutrient solution, in super clean bench, aseptic glue is scraped or cotton swab stretches in bottle ware, under the visual inspection, strike off unmarked space;
3) clean the cell that eccysis is wiped off with the PBS scavenging solution;
4) inject nutrient solution and continue to cultivate, still have inoblast residual, can repeat to strike off till remove fully as finding.
Described a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth is characterized in that described purification process adopts the digestion exclusive method, and its processing step is:
1) at first with the mixture slaking liquid rinsing culturing cell that contains 0.25% trypsinase and 0.02%EDTA, makes it the submergence cell, discard Digestive system then, make cell be in thin layer Digestive system digestion state, under inverted microscope, regularly observe also wave and culture bottle frequently;
2) come off when seeing the half inoblast, after the cell around the tumour cell circle was web-like, adding immediately contained the serum nutrient solution and stops digestion, softly shakes, blows and beats the card cell afterwards inoblast is come off;
3) with the soft rinsing of PBS scavenging solution, Digestive system and the floating cell of trip are discarded fully, in former bottle, add new nutrient solution after the re-treatment and continue to cultivate.
Described a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth, it is characterized in that cell grows to desired number after, adopt the mixture slaking liquid digestion of 0.25% pancreatin-0.02%EDTA and collect tumour unicellular.
Described a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth is characterized in that described nutrient solution is a 15-20% foetal calf serum nutrient solution.
Described a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth is characterized in that described collagenase digesting liquid is the mixed solution that contains collagenase lmg/ml and Unidasa 0.5mg/ml, filters to be dissolved in the DMEM nutrient solution after aseptic.
Described a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth is characterized in that described nutrient solution is for containing 10-15% foetal calf serum nutrient solution.
The conventional former breeding method of being commissioned to train is cultivated the large bowel cancer tumour cell of good order and condition, success ratio very low (6%), the former breeding method of being commissioned to train of the present invention can reach more than 33%, significantly improved former generation colorectal cancer cells short-term vitro culture probability of successful, and it is simple for process, cost is low, for the experimental study of using term growth colorectal cancer cells of former generation provides an alternative good platform.
Embodiment
Embodiment 1:
(1) Operation theatre obtains the fresh state tumor specimen; After stroke-physiological saline solution was cleaned and cut off filth and necrotic zone, Iodophor soaked, jog was sterilized 5 minutes; The PBS scavenging solution that contains two anti-(1000U/ml penicillin and streptomycins) cleans twice; Be transferred in the clean glass dish after the sterilization.
(2) use aseptic eye scissors tweezer with tissue block be cut into as far as possible the fine tissue piece (＜1mm); Insert in the 15ml centrifuge tube and staticly settle, remove supernatant, clean with the PBS scavenging solution once more afterwards, supernatant is removed in centrifugation.
(3) be transferred in the 50ml centrifuge tube, press 1: 10 adding about 10-15ml collagenase digesting liquid (collagenase 1mg/ml+ Unidasa 0.5mg/ml) according to the tissue block total amount, place 37 ℃ of water-baths to shake the case effect 0.5-3 hour, can be 0.5 hour, 1 hour, 2 hours or 3 hours according to the concrete time of actual digestible degree; The collagenase addition also can be 1: 8 to 1: 12.
(4) wait to manage and stop digestion when inner tissue's piece all is half batting shape or fine particle shape, if digestion excessively, it is unicellular to adopt 100 order steel sieve to filter, and obtains the remaining tumour gland group in screen cloth top, is collected into and leaves standstill 2 minutes in the centrifuge tube, and dropper is inhaled and abandoned supernatant liquor.
(5) use the resuspended and flushing of serum-free DMEM nutrient solution, supernatant liquor is abandoned in centrifugation; It is resuspended to add serum-free DMEM nutrient solution afterwards once more, and supernatant liquor is abandoned in cleaning, centrifugal.
(6) add 20% foetal calf serum nutrient solution 4ml, mixing, with dropper little tissue block and body of gland particle are inserted respectively in two the six orifice plate plate holes or 6cm culture plate in, shake gently it be evenly distributed.
(7) cultivate after 48 hours, discard original fluid, contain 10% foetal calf serum nutrient solution along cultivating wooden partition adding capacity.
(8) continue to cultivate observe, wait for that cell grows out around small tissue blocks, changed liquid once in per 3 days, observation of cell circle size therebetween, and be carried out to the fibrocyte removing as follows according to the fibroblastic growth situation with asing one sees fit.
(9) to the tumour cell coverage rate be suitable size and cell when being unlikely to overstocked (cell extend to outside the tissue block about 10 layers), mixture slaking liquid appropriateness peptic cell with 0.25% pancreatin-0.02%EDTA, softly blow down free cell and collect and centrifugation, resuspended, be inoculated in and proceed to cultivate or directly carry out applied research in other culture hole or the culture dish.
Conclusion: through facts have proved in a large number, in the former process of supporting of being commissioned to train of colorectal cancer cells, single primary tumor cell is difficult in external existence.Have only agglomerating tumour cell or tumour gland group just can grow in the vitro culture environment.The used method principle of this law mainly is to make tissue block become tiny by the physics shearing action, and the piece inner tumour cell is mainly existed with glandule unity structure.The matter composition is come by digestion and helps outside nutrient solution of tumor epithelial cell cells contacting and culture dish bottom between around suitably collagenase digesting makes tumour gland group.Because of mesenchyma stroma of tumors to the collagenase sensitivity, essence is insensitive, so tumor epithelial cell is easier to expose and exist with more complete gland unity structure after digesting, is more conducive to growth of tumour cell.
Normal and the tumour cell of inoblast mixes growth simultaneously, causes to be difficult to the purifying tumour cell.And inoblast often gets soon than growth of tumour cell, finally can suppress the growth of tumour cell.Therefore get rid of inoblast and become key in the tumor cell culture.The fibroblastic method of described eliminating comprises mechanical curettage method and digestion exclusive method, can separately or unite use.
The mechanical curettage method:
1) mark: microscopically is observed, with the position of marker pen growth tumour cell under the circle of the back side of culturing bottle, ware;
2) strike off: discard nutrient solution, in super clean bench, aseptic glue is scraped or cotton swab stretches in bottle ware, under the visual inspection, strike off unmarked space;
3) clean twice with the PBS scavenging solution, the cell that eccysis is wiped off;
4) inject nutrient solution and continue to cultivate, still have inoblast residual, can repeat to strike off till remove fully as finding.
The digestion exclusive method:
Cardinal principle is: the former foster tumour cell of being commissioned to train is blunt to the susceptibility of trysinization liquid, and inoblast is comparatively responsive, so two class cell detachment time phase differences are more.Specific procedure is:
1) at first uses 0.25% trypsinase and 0.02%EDTA (1: 1) mixture slaking liquid rinsing culturing cell once, make it the submergence cell, discard Digestive system then, make cell be in thin layer Digestive system digestion state, and regularly observation and wave and culture bottle frequently under inverted microscope.
2) split away off when seeing the half inoblast, after the cell around the tumour cell circle is web-like, just adds immediately and contain the foetal calf serum nutrient solution and stop digestion, softly shake, blow and beat the card cell afterwards inoblast is come off.
3) the soft diafiltration of PBS scavenging solution discards Digestive system and the floating cell of trip fully, adds new nutrient solution and continue to cultivate in former bottle.After this method processing, inoblast comes off earlier than tumour cell is easy.Through repeated treatments several times, can be most inoblast Ex-alls.
Embodiment 2:
(1) Operation theatre obtains the fresh and alive position of tumour sample; After stroke-physiological saline solution is cleaned and is cut off filth and necrotic zone, Iodophor soaking disinfection 5 minutes; The PBS that contains two anti-(1000U/ml penicillin and streptomycins) cleans twice; Be transferred in the clean glass dish in sterilization back.
(2) use aseptic eye scissors tweezer that tissue block is cut into fine tissue piece (average 1--2mm) (unsuitable meticulous, otherwise organize easily floating).
(3) insert centrifugation in the 15ml centrifuge tube; Abandon supernatant liquor with the dropper suction, use the resuspended and flushing once more of PBS scavenging solution, centrifugation is inhaled and is abandoned supernatant liquor; It is resuspended to add serum-free DMEM nutrient solution afterwards, cleans, and centrifugation is inhaled and abandoned supernatant liquor.
(4) add an amount of collagenase digesting liquid (collagenase 1mg/ml+ Unidasa 0.5mg/ml) according to the tissue block amount, place 37 ℃ of water-baths to shake the case effect 30 minutes, make matter separation between the tissue block surface, expose tumour gland group; Serum-free DEME nutrient solution cleans afterwards, precipitates 1 minute, the not centrifugal supernatant liquor of directly abandoning.Clean the centrifugal supernatant liquor of abandoning with serum-free DEME nutrient solution once more.
(5) add the pure foetal calf serum of about 0.5-1.0ml (FBS), mixing.
(6) plantation of tissue block: the dropper big slightly with bore is drawn to the tissue block that pure foetal calf serum (FBS) soaks in culturing bottle or the culture dish, with tissue block dial to density suitably, be evenly distributed, evenly drip pure foetal calf serum to covering culturing bottle or culture dish bottom surface fully, then culturing bottle or culture dish slowly being tilted to place amasss at the bottom of bottle serum, heeling condition is put into incubator, and be in tissue block and stick the adherent phase this moment.
(7) after 6-12 hour, add 10-15% foetal calf serum nutrient solution, tissue block just is submerged and is unlikely to float, horizontality is reentered in the incubator and cultivates.
(8) change liquid:, must change liquid in 24-48 hour 1 time in order to keep enough nutrition and acid base equilibriums.Before guaranteeing under the axial situation of culturing bottle that the old nutrient solution that sucking-off is whole adds the new nutrient solution of isodose along the bottle wall, and guarantees that nutrient solution can just cover the tissue block surface when changing liquid several times.This is adherent phase trace culturing process.Total incubation time of adherent phase trace nutrient solution generally will keep 3 days or longer, and the speed that grows cell on tissue block is decided.Treat to grow around the tissue block than many cells and adherent after can be considered and enter cell vegetative period, nutrient solution could be increased to convention amount or more when changing liquid, and change the static cultivation that liquid should keep 2-4 days at every turn, be beneficial to the ramp of cell.
(9) continue to cultivate observe, wait for that cell grows out around small tissue blocks, change observation of cell circle size during the liquid, take the circumstances into consideration to be carried out to fibrocyte according to the fibroblastic growth situation and remove by embodiment 1 described same procedure.For the tissue block of having only inoblast to climb out of, it is struck off fully.
(10) harvested cell: after treating that cell growth reaches the quantity of requirement of experiment, digest to tissue block with the mixture slaking liquid of 0.25% pancreatin-0.02%EDTA and to come off.Add and contain serum nutrient solution piping and druming evenly and to collect tumour unicellular.Look the activity and the requirement of experiment of tissue block, but the sucking-off tissue block continues to cultivate by present method.Experimental requirement, collected cell can being done goes down to posterity cultivates or cell is used for subsequent experimental research.
Conclusion: the culture method of cell attachment by force that present embodiment is set up is grown tumour cell for the good tumor tissues of drawing materials is very easy.Tissue mass cell culture relatively in the past, can reduce tissue block adherent difficulty and cell injury and cause climb out of difficulty.This culture method characteristics are: tissue block adds micro-nutrient solution after for some time is sticked in the culture plate bottom surface of the clear lining of pure blood.Change liquid in timing and keep under the well-fed situation, tissue block is not floated for a long time, and good environment has been created in growth to cell attachment, has increased the former foster success ratio of being commissioned to train of tissue block.
Claims (10)
1. former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth is characterized in that comprising following processing step:
1) the fresh state tumor specimen is obtained in operation, and after physiological saline cleaned and cuts off filth and necrotic zone, Iodophor soaking disinfection 5 minutes adopted to contain two anti-PBS scavenging solutions and clean;
2) in containing two anti-PBS scavenging solutions above-mentioned tumor specimen is cut into the fine tissue piece, leaves standstill, go supernatant liquor, clean with containing two anti-PBS scavenging solutions more afterwards, supernatant liquor is removed in centrifugation;
3) employing collagenase digestion or tissue block adherent method are organized adherent in nutrient solution and cell climbs out of, and described nutrient solution is a 10-20% foetal calf serum nutrient solution;
4) in above-mentioned nutrient solution, continue to cultivate and purification process, keep cell and grow to desired number.
2. a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth as claimed in claim 1 is characterized in that described collagenase digestion processing step is:
1) according to specimen block amount, by 1: the volume ratio of 8-12 adds collagenase digesting liquid, places 37 ℃ of water-baths to shake case effect 0.5-3 hour, all is half batting shape or fine particle shape to tissue block, leaves standstill, and supernatant liquor is abandoned in the dropper suction;
2) centrifugation is abandoned supernatant liquor with the dropper suction, uses resuspended, the purge mixing of serum-free DMEM nutrient solution then, and is centrifugal, abandons supernatant liquor;
3) in 10-20% foetal calf serum nutrient solution, after 48 hours, discard original fluid, change to the new nutrient solution of capacity, and changed one time nutrient solution in per 3 days with thin quantity of fluid training method cultivation;
3. a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth as claimed in claim 1 is characterized in that described tissue block adherent method processing step is:
1) sample tissue after treatment adds resuspended, the cleaning of serum-free DMEM nutrient solution, and supernatant liquor is abandoned in centrifugation;
2) according to specimen block's amount, by 1: the volume ratio of 8-12 adds collagenase digesting liquid, places 37 ℃ of water-baths to shake the case effect 20-40 minute, make matter separation between the tissue block surface, expose tumour cell, clean with serum-free DEME nutrient solution afterwards, staticly settle, abandon supernatant liquor;
3) add pure foetal calf serum (FBS), mixing fully soaks into the sample tissue;
4) plantation of tissue block: the tissue block that pure foetal calf serum (FBS) is soaked is drawn in culturing bottle or the culture dish, be evenly distributed, continue to drip pure foetal calf serum (FBS) to covering culturing bottle or culture dish bottom surface fully, culturing bottle or the slow heeling condition of culture dish are placed in the incubator, and it is adherent that tissue block is sticked;
5) after 6-12 hour, add 10-20% foetal calf serum nutrient solution, tissue block just is submerged and is unlikely to float, the level position is put into incubator and is cultivated again, how much changes one time nutrient solution every 24-48 hour according to tissue block.To tissue block adherent firmly after, add the capacity nutrient solution and changed liquid once in per 72 hours.
4. a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth as claimed in claim 1 is characterized in that described purification process adopts the mechanical curettage method, and its processing step is:
1) mark: microscopically is observed, with the position of marker pen growth tumour cell under the circle of the back side of culturing bottle or culture dish;
2) strike off: discard nutrient solution, in super clean bench, aseptic glue is scraped or cotton swab stretches in bottle ware, under the visual inspection, strike off unmarked space;
3) clean the cell that eccysis is wiped off with the PBS scavenging solution;
4) inject nutrient solution and continue to cultivate, still have inoblast residual, can repeat to strike off till remove fully as finding.
5. a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth as claimed in claim 1,
It is characterized in that described purification process adopts the digestion exclusive method, its processing step is:
1) at first with the mixture slaking liquid rinsing culturing cell that contains 0.25% trypsinase and 0.02%EDTA, makes it the submergence cell, discard Digestive system then, make cell be in thin layer Digestive system digestion state, under inverted microscope, regularly observe also wave and culture bottle frequently;
2) come off when seeing the half inoblast, after the cell around the tumour cell circle was web-like, adding immediately contained the serum nutrient solution and stops digestion, softly shakes, blows and beats the card cell afterwards inoblast is come off;
3) with the soft rinsing of PBS scavenging solution, Digestive system and the floating cell of trip are discarded fully, in former bottle, add new nutrient solution after the re-treatment and continue to cultivate.
6. a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth as claimed in claim 1, it is characterized in that cell grows to desired number after, adopt the mixture slaking liquid digestion of 0.25% pancreatin-0.02%EDTA and collect tumour unicellular.
7. a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth as claimed in claim 2 is characterized in that described nutrient solution is a 15-20% foetal calf serum nutrient solution.
8. a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth as claimed in claim 2, it is characterized in that described collagenase digesting liquid is the mixed solution that contains collagenase 1mg/ml and Unidasa 0.5mg/ml, filter and be dissolved in the DMEM nutrient solution after aseptic.
9. a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth as claimed in claim 3 is characterized in that described nutrient solution is for containing 10-15% foetal calf serum nutrient solution.
10. a kind of former breeding method of being commissioned to train of keeping large bowel cancer tumour cell term growth as claimed in claim 3, it is characterized in that described collagenase digesting liquid is the mixed solution that contains collagenase 1mg/ml and Unidasa 0.5mg/ml, filter and be dissolved in the DMEM nutrient solution after aseptic.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103865876A (en) * | 2014-03-26 | 2014-06-18 | 西北民族大学 | Method for primary culture of tumor cells |
| CN106047813A (en) * | 2016-07-11 | 2016-10-26 | 广西医科大学 | Rapid extraction method of fibroblast and application |
| CN106479891A (en) * | 2016-12-20 | 2017-03-08 | 北京市肿瘤防治研究所 | Colorectal cancer primitive cell culture kit and its application process |
| CN110777116A (en) * | 2019-10-11 | 2020-02-11 | 纳肽得有限公司 | Tumor sample pretreatment method |
| CN110846280A (en) * | 2019-12-13 | 2020-02-28 | 北京和合医学诊断技术股份有限公司 | Primary human intestinal cancer cell and culture method and application thereof |
| CN110904043A (en) * | 2019-12-13 | 2020-03-24 | 北京和合医学诊断技术股份有限公司 | Primary cells of human intestinal cancer, application and culture method |
| CN111004782A (en) * | 2019-12-13 | 2020-04-14 | 北京和合医学诊断技术股份有限公司 | Primary human intestinal cancer cell and culture method and application thereof |
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| CN1301327C (en) * | 2003-09-25 | 2007-02-21 | 中山大学肿瘤防治中心 | Human Tumor cell separating and purifying process |
| CN1252259C (en) * | 2004-04-20 | 2006-04-19 | 南开大学 | Sub-clone cell line mestastatic-human liver cancer and establishing method |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865876A (en) * | 2014-03-26 | 2014-06-18 | 西北民族大学 | Method for primary culture of tumor cells |
| CN103865876B (en) * | 2014-03-26 | 2016-06-01 | 西北民族大学 | A kind of method of tumour cell original cuiture |
| CN106047813A (en) * | 2016-07-11 | 2016-10-26 | 广西医科大学 | Rapid extraction method of fibroblast and application |
| CN106479891A (en) * | 2016-12-20 | 2017-03-08 | 北京市肿瘤防治研究所 | Colorectal cancer primitive cell culture kit and its application process |
| CN110777116A (en) * | 2019-10-11 | 2020-02-11 | 纳肽得有限公司 | Tumor sample pretreatment method |
| CN110846280A (en) * | 2019-12-13 | 2020-02-28 | 北京和合医学诊断技术股份有限公司 | Primary human intestinal cancer cell and culture method and application thereof |
| CN110904043A (en) * | 2019-12-13 | 2020-03-24 | 北京和合医学诊断技术股份有限公司 | Primary cells of human intestinal cancer, application and culture method |
| CN111004782A (en) * | 2019-12-13 | 2020-04-14 | 北京和合医学诊断技术股份有限公司 | Primary human intestinal cancer cell and culture method and application thereof |
| CN111004782B (en) * | 2019-12-13 | 2021-08-17 | 北京和合医学诊断技术股份有限公司 | Primary human intestinal cancer cell and culture method and application thereof |
| CN110846280B (en) * | 2019-12-13 | 2021-08-17 | 北京和合医学诊断技术股份有限公司 | Primary human intestinal cancer cell and culture method and application thereof |
| CN110904043B (en) * | 2019-12-13 | 2021-09-21 | 北京和合医学诊断技术股份有限公司 | Primary cells of human intestinal cancer, application and culture method |
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