CN108070561A - A kind of isolation and culture method of the primary colon cancer cell of people - Google Patents
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Abstract
The present invention provides a kind of isolation and culture methods of the primary colon cancer cell of people, comprise the following steps：(1) fresh colon cancer tissue is cut under aseptic condition, laboratory is transported in 4 DEG C of sealings back；(2) tissue treatment instrument sterilizes；(3) with operating scissors separate colon cancer tissue, will tissue be put into precooling it is sterile containing washed in dual anti-PBS liquid for several times remove blood vessel, fat, slough；(4) tissue is shredded, and is digested respectively with the pancreatin and iv Collagenase Types of preheating；(5) digestion is terminated, supernatant is abandoned in centrifugation；(6) cell filtrate is taken to detect cytoactive with trypan blue staining；(7) blake bottle being inoculated into after cell count after coating；(8) 37 DEG C, 5%CO are placed in2It is cultivated in environment；(9) cell purification；(10) cell secondary culture；(11) cellular morphology is identified；(12) cell survival rate measures；(13) Immunological Identification.A kind of isolation and culture method of human colon cancer cell provided by the invention, easy to operate, gained cell quantity is more, and survival rate is high, is a kind of primary separation of preferable human colon cancer cell and cultural method, and reliable cellular resources are provided for experiment.
Description
Technical field
The invention belongs to technical field of modern biotechnology cell culture, and in particular to a kind of primary colon cancer cell of people
Isolation and culture method.
Background technology
Colon cancer is one of clinical most common malignant tumour and lethal factor, although chemotherapeutics new in recent years is continuous
It emerges in large numbers, but its complete cure rate and long-term survival rate are still very low, the appearance of such case may be related with tumor stem cell.
Research finds that there are a small amount of CD133 in colon cancer+Cell, these cells be able to maintain that colon cancer and colon cancer drug resistance and
It treats closely related.Therefore this kind of stem cell for finding colon cancer has a very important significance for the treatment of colon cancer.
The research of human colon carcinoma at present is mostly using cell line, but long-term subculture in vitro separately culture can make its cell characteristics and egg
White expression changes, and can generate interference to experimental result in human colon carcinoma experimental study, influence to test expected results.Pass through
The human colon cancer cell that original cuiture obtains can be to avoid influence of the cell line Long Term Passages to cell characteristics, and can reduce group
Interference of the interstitial components to experimental result is knitted, reliable data is provided for the research of various experiments.
The human colon cancer cell isolated culture method having built up at present is isolated mostly based on tissue mass cell culture
Cell purity is not high, and quantity is few, and incubation time is longer.On the culture of human colon cancer cell, introduce both at home and abroad at present
Method is unfavorable for practical application there are the problems such as poor repeatability or complexity.It is the present invention is intended to provide a kind of easy to operate, efficient
The method for obtaining growth conditions are good, purity is high human colon cancer cell, establishes that a set of to improve reliable human colon cancer cell external
Culture technique.
The content of the invention
According to the above problem, the present invention provides a kind of isolation and culture method of the primary colon cancer cell of people, this method
Easy to operate, cell yield and survival rate are high, are a kind of primary colon cancer cell cultural methods of ideal people, can meet
The requirement of a variety of bio-chemical characteristics.
The technical solution that the present invention uses is as follows：
(1) fresh colon cancer tissue is cut under aseptic condition, with PBS (concentration is 0.008~0.015M, PH for 7.2~
7.4) rinse 2~3 times, be stored in DMEM/F12 (1: 1) culture medium of 1000U/ml penicillin and 1000mg/L streptomysins, 4
Laboratory is transported in DEG C sealing back.
(2) tissue treatment instrument is soaked in 75% ethanol water of concentration, then is placed in aseptic operating platform ultraviolet sterilization
30min takes out instrument, is dried in aseptic operating platform.
(3) go out tissue with sterile tweezer in super-clean bench, it is sterile containing dual anti-(penicillin of 100u/ml, 100u/ to be put into precooling
The streptomysin of ml) PBS liquid (concentration be 0.008~0.015M, PH be 7.2~7.4) in, culture dish be placed on ice washing remove system
Film, blood vessel, fat, slough.
(4) under aseptic condition, eye scissors is used in the PBS (concentration is 0.008~0.015M, and PH is 7.2~7.4) of precooling
Colon cancer tissue is cut into the fragment of 1mm × 1mm × 1mm, is placed in sterile centrifugation tube.
(5) 10min is digested with the trypsase-EDTA digestive juices of 37 DEG C of preheatings, then is shaken with 37 DEG C of constant temperature of iv Collagenase Types
3~4h of bed digestion.
(6) with 10ml containing 10% hyclone, dual anti-(penicillin of 100u/ml, the streptomysin of 100u/ml), 4~6 μ g/
DMEM/F12 (1: 1) mixed culture medium (abbreviation mixed culture medium) of ml insulin terminates digestion, 200 mesh sieve net filtrations, gained
250~350g of filtrate centrifuges 5min, is repeated twice.
(7) cell filtrate is taken to detect cytoactive with trypan blue staining.
(8) add in complete medium to be resuspended, cell counting count board counts.
(9) inoculating cell is placed in 37 DEG C, 5%CO to spreading in the coated blake bottle of poly-D-lysine2It is cultivated in environment,
Liquid is changed after for 24 hours for the first time, hereafter 2~3d is changed the liquid once.
(10) cell purification：When primary cell adherent growth is up to 80%, 0.25% pancreatin is added to digest, observes into fiber finer
Born of the same parents are rounded, and when partial exfoliation adds in DMEM/F12 (1: the 1) culture mediums of 2ml containing 10% hyclone and terminates digestion, removal immediately
The fibroblast to come off and the cancer cell of poor activity.
(11) cell secondary culture：Colon cancer cell discards culture medium when growing after purification to 80%~90%, (dense with PBS
Spend for 0.008~0.015M, PH is 7.2~7.4) washing 2~3 times, with 0.08% 3~5min of Trypsin Induced, it is inverted
Micro- Microscopic observation, when cell takes off wall, retraction is rounded, and mixed culture medium is added in when diopter reduces and terminates digestion, uses suction pipe
It gently blows and beats, cell suspension is made, passed in 1: 3 ratio, be denoted as P1。
(12) cellular morphology observation identification, starts after 16h adherent, and Microscopic observation cell is in fusiformis or polygon, nucleus
Centrally located, in oval, kytoplasm protruding protrusion different in size starts rapid multiplication after 3d, after 7d cell can grow to
More than 80%, pencil arrangement, cell homogeneous clear, cell space is limpid, and refractivity is good, is dyed with auspicious lucky decoration method and observes cell shape
State.
(13) colon cancer cell Immunological Identification takes P1For cell, cell angle egg is detected respectively with immunocytochemical method
White and Carcinoembryonic Antigen CEA.
Wherein, PBS 4 DEG C of precoolings described in step (3) (4), PBS concentration are 0.01M, and PH is 7.2~7.4, culture dish
It is placed in aseptic operating platform on ice, is in order to which tissue is made to be in sterile, low temperature environment.
Wherein, step (5) described digestive environments are 37 DEG C, 5%CO2Incubator, 0.1%~0.2% iv Collagenase Types
Digest the Trypsin Induced 10min of 3.5h and 0.2%~0.3%.
Wherein, the trypsase used in step (5) containing 0.01% EDTA with the Ca in absorptive tissue2+、Mg2+, thus into
One step promotes cell separation.
Wherein, step (6) described culture medium is DMEM/F12 (1: 1) culture medium, containing 10% hyclone, 100u/ml's
Penicillin, 100u/ml streptomysin dual anti-, the insulin of 5 μ g/ml.
Wherein, in step (10) described purification process, digestion is terminated immediately when fibroblast completely falls off；
Wherein, when colon cancer cell bounces back when step (11) cell passes on, mixing should be added in immediately by tending to change bowlder
Culture medium terminates digestion.
Advantageous effect
The present invention establishes a kind of simple, efficient method for being separately cultured the primary colon cancer cell of people, and gained is primary thin
Born of the same parents are through morphological observation and identification：Quantity is more, purity is high, activity is good, and up to more than 95%, cell purity reaches cell survival rate
More than 96%.
The present invention is using pancreatin and clostridiopetidase A simultaneous digestion method, better than mechanical phonograph recorder separation and tissue block method, the cell of acquisition
Quantity is more, and cellular damage is small, cell purity higher.Two kinds of enzymes digest respectively makes tissue fully be digested, to the damage of cell
Wound is preferably minimized, and is conducive to that colon cancer cell is adherent and late growth.Obtained colon cancer cell purity is 80% or so, then passes through
Trypsin Induced purifying is crossed, cell purity is up to more than 96%, and present invention gained colon cancer cell quantity is more, stable quality, again
Renaturation is good.
Compared with mechanical phonograph recorder separation and tissue block method, cell is evenly stronger obtained by collagenase digestion is attached to
Bottle wall is cultivated, enhances its viability.Mechanical phonograph recorder separation easily makes cell damage.Tissue block method is easy to operate, at low cost, but thin
Born of the same parents' survival rate is relatively low.
The present invention is coated with blake bottle using poly-D-lysine, and cell growth state is good, can meet the primary colon cancer cell of people
The requirement of experiment, poly-D-lysine coating make simpler, easily preservation；This method is economical and practical, simple and easy to do, is conducive to build
Vertical in vitro models cell studies the characteristic of colon cancer cell, provides reliable cellular resources for subsequent experiment, and is
The research that clinic carries out the variation of different type colon cancer cell functional structure and treatment etc. provides basis.
Description of the drawings
The human colon cancer cell picture (200 ×) of Fig. 1 cultures 2d
The human colon cancer cell picture (200 ×) of Fig. 2 cultures 3d
The human colon cancer cell picture (200 ×) of Fig. 3 cultures 7d
The human colon cancer cell picture (200 ×) of Fig. 4 cultures 14d
Specific embodiment
Used term in the present invention unless otherwise indicated, generally there are those of ordinary skill in the art usually to manage
The meaning of solution.
The present invention is described in detail with example below in conjunction with the accompanying drawings, and protection content of the invention is not limited to following implementations
Example.
Below in an example, the various processes and method not being described in detail are conventional methods as known in the art.
This experiment laboratory apparatus used and reagent are as follows：
Surgical operating instrument is a set of, CCK-8 cell counts box (Sigma, the U.S.), enzyme-linked immunosorbent assay kit (R＆D,
The U.S.), inverted microscope (XDS-1A, Shanghai), fluorescence microscope (Leica, the U.S.), refrigerated centrifuge (TD24B-WS, on
Sea), ultra low temperature freezer (middle U.S. of section water chestnut), liquid-transfering gun (Eppendorf, the U.S.), electronic analytical balance (Sartorius, the U.S.),
Superclean bench (HJ-CJ-1D, Shanghai), CO2Cell incubator (SANYOMCO-17AI, Japan).
DMEM/F12 (1: 1) is purchased from Corning companies, EDTANa2Be purchased from Sigma companies, Pen .- Strep it is dual anti-in
Gibco is bought, and hyclone purchase is in Bioind companies, and poly-D-lysine purchase is in Gibco companies of the U.S., PBS self-controls
(8.0gNal、0.2gKCl、0.2gKH2PO4、1.44gNa2HPO4It being dissolved in 1000ml deionized waters, adjustment PH is 7.2~7.4,
No special to point out, the PBS used in this patent is all formulated therefore), iv Collagenase Types and trypsase purchase are public in German Serva
Department, Tissue Culture Flask, centrifuge tube are purchased from Corning companies of the U.S., and trypan blue is bought in U.S. Biofer, diaminobenzidine
(DAB) purchase of colour reagent box is in Boster.
Technical solution provided by the invention includes the following steps：
1. cut fresh colon cancer tissue under aseptic condition, with PBS (concentration is 0.008~0.015M, PH for 7.2~
7.4) rinse 2~3 times, be stored in DMEM/F12 (1: 1) culture medium of 1000U/ml penicillin and 1000mg/L streptomysins, 4
Laboratory is transported in DEG C sealing back.
2. tissue treatment instrument is soaked in 75% ethanol water of volume fraction, then it is placed in aseptic operating platform ultraviolet sterilization
30min takes out instrument, is dried in aseptic operating platform.
3. going out tissue with sterile tweezer in super-clean bench, it is sterile containing dual anti-(penicillin of 100u/ml, 100u/ml to be put into precooling
Streptomysin) PBS liquid (and concentration be 0.008~0.015M, PH be 7.2~7.4) in, culture dish be placed on ice washing remove system
Film, blood vessel, fat, slough.
4. under aseptic condition, eye scissors is used in the PBS (concentration is 0.008~0.015M, and PH is 7.2~7.4) of precooling
Colon cancer tissue is cut into the fragment of 1mm × 1mm × 1mm, is placed in sterile centrifugation tube.
5. for the trypsase-EDTA digestive juices of the 0.25g/l preheated with 37 DEG C in 37 DEG C, CO2 incubators digest 10min,
Again with 37 DEG C of constant-temperature table (150r/min) digestion 3.5h of iv Collagenase Types of iv Collagenase Types 0.1g/l, collagenase digesting process
In every 30min piping and druming once.
6. 10ml is added in containing 10% hyclone, dual anti-(penicillin of 100u/ml, the streptomysin of 100u/ml), 5 μ g/ml
Insulin, DMEM/F12 (1: 1) mixed culture medium (abbreviation mixed culture medium) that pH is 7.4 terminate digestion, gained filtrate 300g
5min is centrifuged, removes supernatant, retains precipitation, is repeated twice.
7. cell filtrate is taken to detect cytoactive with trypan blue staining：Trypan Blue exclusion assay draws 0.2ml P1In generation, is thin
Born of the same parents' suspension adds in 0.4% trypan blue solution of 0.2ml, and piping and druming is uniform, then draws a small amount of suspension along cell counting count board upper cover plate
Edge slowly instills, until just being observed under cover plate full of suspension under inverted microscope, is prompted if nucleus is not colored thin
Born of the same parents are survived, if nucleus is blue by dye, prompting cell death, the four big gitter cell number of counting (four big lattice total number of cells/4 ×
104), calculate cell survival rate：Total viable cell/(total viable cell+dead cell sum) × 100%.
7. adding in complete medium to be resuspended, cell counting count board counts, and is placed in cell counting count board center and counts dedicated lid
Slide, with glass siphon draw cell, allow siphon pipe on the cover slip or the tally of downside recessed Bad place outflow suspension, until cover
Until slide is liquid filled, the total number of cells counted under microscope in the block plaid of corner is put.It is only counted for the cell of crimping
It is reaching the standard grade and left line person, is being counted for cell mass by individual cells, tetra- big lattice total number of cells/4 × 10 of cell number/mL=4。
8. add culture medium after the completion of counting is adjusted to 1 × 10 by cell density5A/ml, inoculating cell rely to poly is spread
In the coated blake bottle of propylhomoserin, 37 DEG C, 5%CO are placed in2It is cultivated in environment, changes liquid afterwards for 24 hours, hereafter 2~3d is changed the liquid once.
9. cell purification：When primary cell adherent growth is up to 80%, 0.25% pancreatin is added to digest to cell rounding, into fiber
DMEM/F12 (1: the 1) culture mediums of 2ml containing 10% hyclone is added to terminate digestion during cell detachment, remove come off into fiber finer
Born of the same parents and the cancer cell of poor activity.
10. cell secondary culture：It can be passed on when colon cancer cell is continuously in blocks, discard mixed culture medium, use PBS
(concentration is 0.008~0.015M, and PH is 7.2~7.4) washing 2~3 times, with 0.08% 3~5min of Trypsin Induced, falls
Micro- Microscopic observation is put, when cell takes off wall, retraction is rounded, and mixed culture medium is added in when diopter reduces and terminates digestion, with suction
Pipe is gently blown and beaten, and cell suspension is made, is passed in 1: 3 ratio, is denoted as P1, later same method passed on.
11. cell survival rate measures：Trypan Blue exclusion assay draws 0.2ml P1Add in 0.2ml's for cell suspension
0.4% trypan blue solution, piping and druming is uniform, then draws a small amount of suspension and is slowly instilled along cell counting count board upper cover plate edge, until lid
Under piece just full of suspension, is observed under inverted microscope, cell survival is prompted if nucleus is not colored, such as nucleus quilt
Dye is blue, prompts cell death, counts four big gitter cell number (four big lattice total number of cells/4 × 104), calculate cell survival rate：
Total viable cell/(total viable cell+dead cell sum) × 100%.
The observation identification of 12 cellular morphologies, starts after 16h adherent, and Microscopic observation cell is in fusiformis or polygon, nucleus position
In center, in oval, kytoplasm protruding protrusion different in size starts rapid multiplication after 3d, after 7d cell can grow to
More than 80%, pencil arrangement, cell homogeneous clear, cell space is limpid, and refractivity is good, is dyed with auspicious lucky decoration method and observes cell shape
State.
13. colon cancer cell immunohistochemistry is identified：Take P1For cell, orifice plate is rinsed with PBS, 4 DEG C of fixers are fixed
10min, 1%H2O210~15min is incubated at room temperature, PBS is rinsed 3 times, and confining liquid is added dropwise and is incubated at room temperature 10~15 minutes, is added in multiple
4 DEG C of 10~15min of incubation of digestion working solution are closed, abandon confining liquid, 37 DEG C of incubation 1h of primary antibody are added dropwise, are added dropwise specific biotinylated two
Peroxide complex enzyme is added dropwise in anti-37 DEG C of incubations 30min, PBS after rinsing 3 times, DAB color developing agents room is added dropwise in 37 DEG C of incubation 30min
Temperature is protected from light 3~5min of colour developing, and haematoxylin redyes 2~3min after tap water fully rinses, and washing terminates reaction, after differentiation liquid differentiation
With oil blackeite liquid oil blackeite 5min, Microscopic observation result is dehydrated.
300 cells are counted under high-power microscope, human colon cancer cell purity is more than 96%.
The present invention is obtained, and human colon cancer cell quantity is more, and for cell survival rate up to more than 95%, cell purity reaches P1
More than 96%.
The separating obtained human colon cancer cell of the present invention can be largely proliferated, and can be subsequent with 4~6 generation of original cuiture
Experiment provides reliable cellular resources.
The above is only the preferable embodiment of the present invention, but protection scope of the present invention is not limited thereto implementation
Example.Any to make any modification within the spirit and principles in the present invention, equivalent substitution and improvement etc. should be included in this hair
In bright protection domain.
Claims (8)
- A kind of 1. isolation and culture method of the primary colon cancer cell of people, which is characterized in that comprise the following steps：(1) fresh colon cancer tissue is cut under aseptic condition, laboratory is transported in 4 DEG C of sealings back；(2) tissue treatment instrument is soaked in 75% ethanol water of concentration, then is placed in aseptic operating platform ultraviolet sterilization 30min, Instrument is taken out, is dried in aseptic operating platform；(3) tissue is put into precooling is sterile containing in dual anti-PBS liquid, culture dish be placed in washing on ice remove the mesentery on surface, blood vessel, Colon cancer tissue is cut into 1mm × 1mm × 1mm's under aseptic condition by fat, slough in the PBS of precooling with eye scissors Fragment；(4) trypsase-EDTA digestive juices and clostridiopetidase A preheated with 37 DEG C digests respectively；(5) digestion, gained filtrate are terminated with containing hyclone, dual anti-, insulin mixed culture medium (abbreviation mixed culture medium) 250~350g centrifuges 5min, removes supernatant, retains precipitation, is repeated twice；(6) cell filtrate is taken to detect cytoactive with trypan blue staining；(7) add in complete medium to be resuspended, cell counting count board counts；(8) inoculating cell to spreading in the coated blake bottle of poly-D-lysine, is placed in 37 DEG C, 5%CO after cell count2In environment Continue to cultivate, every 2~3d changes a not good liquor；(9) cell purification：Enzyme digestion；(10) cell secondary culture；(11) cellular morphology and growing state observation；(12) cellular immunology is identified.
- 2. the isolation and culture method of human colon cancer cell according to claim 1, it is characterised in that the PBS of precooling used Concentration is 0.01M, and PH is 7.2~7.4.
- 3. the isolation and culture method of human colon cancer cell according to claim 1, it is characterised in that step (4) is described to disappear It is 37 DEG C to change environment, 5%CO2Incubator, with 0.2%~0.3% 5~15min of Trypsin Induced, then with 0.1%~ 0.2% iv Collagenase Types digest 3~4h.
- 4. the isolation and culture method of human colon cancer cell according to claim 3, it is characterised in that described 0.25% Tryptic digestive juice digests 10min, then digests 3.5min with containing 0.1% iv Collagenase Types.
- 5. the isolation and culture method of human colon cancer cell according to claim 4, it is characterised in that described 0.25% Trypsase contain 0.01% divinyl tetraacethyl disodium (EDTANa2)。
- 6. the isolation and culture method of human colon cancer cell according to claim 1, it is characterised in that described in step (5) Culture medium is dual anti-, 4~6 μ g/ml insulin containing 10% hyclone, penicillin containing 100u/ml and 100u/ml streptomysins DMEM/F12 (1: 1) culture medium, pH 7.4.
- 7. the isolation and culture method of human colon cancer cell according to claim 1, it is characterised in that described in step (8) 3~4 μ g/cm of blake bottle2Poly-D-lysine coating, condition of culture be 37 DEG C, 5%CO2Incubator.
- 8. the isolation and culture method of human colon cancer cell according to claim 1, it is characterised in that described in step (9) Cell purification method is to be purified with 0.25% Trypsin Induced.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110592019A (en) * | 2018-06-13 | 2019-12-20 | 北京吉尚立德生物科技有限公司 | Dissociation liquid for colorectal cancer solid tumor tissue sample |
| CN112877289A (en) * | 2021-02-24 | 2021-06-01 | 云南省肿瘤医院(昆明医科大学第三附属医院) | Method for promoting intestinal cancer cell proliferation based on CCL20 and CXCL8 |
| CN112980768A (en) * | 2020-12-29 | 2021-06-18 | 刘艳坤 | Method for establishing novel hepatic fibroblast cell strain |
| CN114279795A (en) * | 2020-09-28 | 2022-04-05 | 成都天士力诺唯生物科技有限公司 | Rapid detection system, detection method and application of tissue sample |
| CN114304066A (en) * | 2022-01-04 | 2022-04-12 | 昆明医科大学 | Method for constructing colorectal cancer PDX model |
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| CN114279795A (en) * | 2020-09-28 | 2022-04-05 | 成都天士力诺唯生物科技有限公司 | Rapid detection system, detection method and application of tissue sample |
| CN112980768A (en) * | 2020-12-29 | 2021-06-18 | 刘艳坤 | Method for establishing novel hepatic fibroblast cell strain |
| CN112877289A (en) * | 2021-02-24 | 2021-06-01 | 云南省肿瘤医院(昆明医科大学第三附属医院) | Method for promoting intestinal cancer cell proliferation based on CCL20 and CXCL8 |
| CN114304066A (en) * | 2022-01-04 | 2022-04-12 | 昆明医科大学 | Method for constructing colorectal cancer PDX model |
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