CN108070560A - A kind of isolation and culture method of the primary stomach 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 stomach cancer cell of people, comprise the following steps：(1) tissue treatment instrument sterilizes；(2) stomach organization is separated with operating scissors, tissue is put into that precooling is sterile to remove bloodstain and slough containing being washed in dual anti-PBS liquid for several times；(3) tissue is shredded, and is digested respectively with the IV Collagenase Types enzyme and trypsase of preheating；(4) digestion is terminated, supernatant is abandoned in centrifugation；(5) blake bottle being inoculated into after cell count after coating；(6) 37 DEG C, 5%CO are placed in2It is cultivated in environment；(7) cell purification；(8) cell secondary culture；(9) morphological observation；(10) cell survival rate measures；(11) Immunological Identification.A kind of isolation and culture method of gastric carcinoma cells 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 gastric carcinoma cells 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 stomach cancer cell of people
Isolation and culture method.
Background technology
Stomach cancer is derived from the malignant tumour of gastric epithelial.First of malignant tumour is accounted in China.In China's early stage stomach
The recall rate of cancer is extremely low, is only 10% or so, and the therapeutic effect of advanced gastric carcinoma is poor, and the specific cause of disease is still uncertain so far.
Stomach cancer is prevented and early stage diagnosis and treatment are the key that reduce incidence gastric cancer death.
Gastric carcinoma cell lines have：HGC-27, the KATO-III in Europe；AGS, SNU-1, the HS764T-1 in the U.S.；China
BGC-823、SGC-7901、MGC-803；MKN-1, MKN-28, MKN-45, MKN-74, NMGC-3 of Japan etc..
The research of stomach cancer at present is mostly using cell line, but long-term subculture in vitro separately culture can make its cell characteristics and albumen table
Up to changing, interference can be generated to experimental result in stomach cancer experimental study, influence to test expected results.Pass through original cuiture
The stomach cancer cell of acquisition can be to avoid influence of the cell line Long Term Passages to cell characteristics, and can reduce tissue interstitial components pair
The interference of experimental result provides reliable data for the research of various experiments.
For the gastric carcinoma cells isolated culture method having built up at present mostly based on tissue mass cell culture, isolated is thin
Born of the same parents' purity is not high, and quantity is few, and incubation time is longer.On the culture of gastric carcinoma cells, the method introduced both at home and abroad at present
There are the problems such as poor repeatability or complexity, it is unfavorable for practical application.The present invention is intended to provide a kind of easy to operate, efficient acquisition
The method for the gastric carcinoma cells that growth conditions are good, purity is high is established and a set of improves reliable gastric carcinoma cells Vitro Culture Techniques.
The content of the invention
According to the above problem, the present invention provides a kind of isolation and culture method of the primary stomach cancer cell of people, this method behaviour
Make simply, cell yield and survival rate are high, are a kind of primary stomach cancer cell cultural methods of ideal people, can meet a variety of
The requirement of bio-chemical characteristics.
The technical solution that the present invention uses is as follows：
(1) 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；
(2) fresh just in vitro stomach organization sample is chosen, with PBS (concentration is 0.008~0.015M, and PH is 7.2~7.4)
It rinses and removes surface blood, mucus and slough, be put into and the incubation chamber of ice bag is placed in aseptic culture fluid transports laboratory back；
(3) go out tissue with sterile tweezer, it is sterile containing dual anti-(penicillin of 100u/ml, the strepto- of 100u/ml to be put into precooling
Element) PBS liquid (concentration be 0.008~0.015M, PH be 7.2~7.4) in, culture dish be placed on ice washing remove surface blood
Pipe, slough and interstitial tissue；
(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
Stomach organization is cut into the fragment of 1mm × 1mm × 1mm, is placed in sterile centrifugation tube；
(5) the IV Collagenase Types and trypsase preheated with 37 DEG C digests respectively, and every 10min piping and druming once, digestion is extremely
Tissue block disappears；
(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) add in complete medium to be resuspended, cell counting count board counts；
(8) 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 48h, 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 fibre
DMEM/F12 (1: the 1) culture mediums of 2ml containing 10% hyclone is added to terminate digestion when tieing up cell detachment, remove come off into fiber
The cancer cell of cell and poor activity；
(10) cell secondary culture：Stomach cancer cell discards culture medium when growing after purification to 80%~90%, with PBS (concentration
For 0.008~0.015M, PH is 7.2~7.4) it washs 2~3 times, with 0.08% 3~5min of Trypsin Induced, it is inverted aobvious
Micro- Microscopic observation, when cell takes off wall, retraction is rounded, and mixed culture medium is added in when diopter reduces and terminates digestion, light with suction pipe
Featheriness is beaten, and cell suspension is made, and is passed in 1: 3 ratio, is denoted as P1；
(11) cellular morphology is observed, and adherent up to 50% after 48h, Microscopic observation cell is in fusiformis, paver shape adherent growth, 3d
After start to be proliferated rapidly, cell can be grown to more than 80% after 7d, and cell homogeneous clear, cell space is limpid, and refractivity is good；
(12) measure of cell count and survival rate, Trypan Blue exclusion assay calculate P1 for cell percent living cells；
(13) stomach cancer cell Immunological Identification takes P1For cell, the double dyes of GFAP and P75 are carried out.
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 30~40min of Trypsin Induced of 1~2h and 0.1%~0.2%.
Wherein, the trypsase used in step (5) containing 0.01% divinyl tetraacethyl disodium with the Ca in absorptive tissue2 +、Mg2+, so as to further promote 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 (9) described purification process, digestion is terminated immediately when fibroblast completely falls off；
Wherein, during the passage of step (10) cell when stomach cancer cell bounces back, mixing training should be added in immediately by tending to become bowlder
It supports base and terminates digestion.
Advantageous effect
The present invention establishes a kind of simple, efficient method for being separately cultured the primary stomach cancer cell of people, gained primary cell
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 two kinds of joint enzyme digestions, and the cell quantity of acquisition is more, and cellular damage is small, and cell purity is more
It is high.Two kinds of enzymes digest respectively, and digestion time is short, and tissue is made fully to be digested, and the damage of cell is preferably minimized, is conducive to
Stomach cancer cell is adherent and late growth.Obtained stomach cancer cell purity is purified using Trypsin Induced 80% or so, into
Fibrocyte and the cancer cell of state difference typically precede stomach cancer cell and are digested, so as to fulfill the purifying to stomach cancer cell,
Gained stomach cancer cell quantity of the invention is more, stable quality, reproducible.
Compared with compared to EDTA+DTT methods, cell is evenly stronger obtained by collagenase digestion is attached to blake bottle
Wall enhances its viability.Cell obtained by EDTA+DTT methods is easily separated from blake bottle, and fibroblast is more.
The present invention is coated with blake bottle using poly-D-lysine, and cell growth state is good, and it is real can to meet the primary stomach cancer cell of people
The requirement tested, poly-D-lysine coating are simpler than APES coating making, more easy to maintain；This method is economical and practical, simple and easy to do,
Be conducive to establish in vitro models cell, study the characteristic of stomach cancer cell, reliable cellular resources are provided for subsequent experiment,
And provide basis for the clinical research for carrying out the variation of different type stomach cancer cell functional structure and treatment etc..
Description of the drawings
The gastric carcinoma cells picture (400 ×) of Fig. 1 cultures 1d
The gastric carcinoma cells picture (400 ×) of Fig. 2 cultures 2d
The gastric carcinoma cells picture (200 ×) of Fig. 3 cultures 7d
The gastric carcinoma cells picture (400 ×) of Fig. 4 cultures 7d
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, inverted microscope (XDS-1A, Shanghai), fluorescence microscope (Leica, the U.S.), low temperature
Centrifuge (TD24B-WS, Shanghai), ultra low temperature freezer (middle U.S. of section water chestnut), liquid-transfering gun (Eppendorf, the U.S.), electronic analysis day
Flat (Sartorius, the U.S.), superclean bench (HJ-CJ-1D, Shanghai), CO2Cell incubator (SANYO MCO-17AI, day
This).
P75 antibody is purchased from Sigma companies, and goat anti-rabbit igg is purchased from Sigma companies, and DMEM/F12 (1: 1) is purchased from
Corning companies, EDTANa2Be purchased from Sigma companies, Pen .- Strep is dual anti-to be bought in Gibco, hyclone purchase in
Bioind companies, poly-D-lysine are bought in Gibco companies of the U.S., and Thy1.1 antibody is purchased from Sigma companies, 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.
Technical solution provided by the invention includes the following steps：
1. 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；
2. fresh just in vitro stomach organization sample is chosen, with PBS (concentration is 0.008~0.015M, and PH is 7.2~7.4)
It rinses and removes surface blood, mucus and slough, be put into and the incubation chamber of ice bag is placed in aseptic culture fluid transports laboratory back；
3. going out tissue with sterile tweezer, it is sterile containing dual anti-(penicillin of 100u/ml, the strepto- of 100u/ml to be put into precooling
Element) PBS liquid (concentration be 0.008~0.015M, PH be 7.2~7.4) in, culture dish be placed on ice washing remove surface blood
Pipe, slough and interstitial tissue；
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
Stomach organization is cut into the fragment of 1mm × 1mm × 1mm, is placed in sterile centrifugation tube；
5. with the iv Collagenase Types of the 0.15g/l of 37 DEG C of preheatings in 37 DEG C, CO2Incubator digests 1.5h, IV Collagenase Types
In digestion process every 15min piping and druming once, then with the (0.01%EDTANa of tetraacethyl disodium containing divinyl2) 0.15g/l
Trypsin Induced 35min, Trypsin Induced are blown and beaten once in the process every 10min；
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. 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, liquid is changed after 48h, 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 stomach 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 is passed on；
11. cellular morphology and growing state observation：Adherent up to 50% after 48h, Microscopic observation cell is in fusiformis, and paver shape pastes
Wall is grown, and starts rapid multiplication after 3d, and cell can be grown to more than 80% after 7d, and cell homogeneous clear, cell space is limpid, refractivity
It is good；
12. 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%；
13. stomach cancer cell Immunological Identification, takes P1For cell, the double dyes of GFAP and P75 are carried out.The stomach cancer for cultivating 10d is thin
Culture solution in born of the same parents' blake bottle washes away, and with PBS (concentration is 0.008~0.015M, and PH is 7.2~7.4) washing 2~3 times, adds in
The formaldehyde of volume fraction 10% fixes cell, places 30min at room temperature, washes away formalin, with PBS vibrations washing 3 times, 5min/
It is secondary, 10% normal sheep serum of volume fraction closing heterogenetic antigen 60min is added dropwise in coverslip cell surface, after the completion of closing
Sheep blood serum is suctioned out, is separately added into the first antibody of GFAP and P75, the titre of GFAP first antibodies is anti-for 1: 200, P75 first
Body titre be 1: 50, in 37 DEG C of incubators be incubated 2h or 4 DEG C at overnight, then with PBS vibration washing 3 times, 5min/ times, after blotting
It is that 60min is incubated at 1: 400,37 DEG C to add in anti-rabbit and anti-mouse fluorescent second antibody, titre, then washs 3 with PBS vibrations
It is secondary, 5min/ times, blot rear gelatin sealing.
300 cells are counted under high power, gastric carcinoma cells purity is more than 96%.
The present invention is obtained, and gastric carcinoma cells quantity is more, P1For cell survival rate up to more than 95%, cell purity reaches
More than 96%.
The separating obtained gastric carcinoma cells of the present invention can be largely proliferated, and can be subsequent reality with 3~5 generation of original cuiture
It tests and reliable cellular resources is provided.
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 stomach cancer cell of people, which is characterized in that comprise the following steps：(1) 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；(2) tissue is put into precooling is sterile containing in dual anti-PBS liquid, and culture dish is placed in washing on ice and removes the blood vessel on surface, necrosis Stomach organization is cut into the broken of 1mm × 1mm × 1mm under aseptic condition by tissue and interstitial tissue in the PBS of precooling with eye scissors Block；(3) clostridiopetidase A and the trypsase of the tetraacethyl disodium containing divinyl preheated with 37 DEG C digests respectively, is blown and beaten every 10min Once；(4) 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；(5) 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；；(6) cell purification：Enzyme digestion；(7) cell secondary culture；(8) cellular morphology and growing state observation；(9) measure of cell survival rate；(10) stomach cancer cell Immunological Identification.
- 2. the isolation and culture method of gastric carcinoma cells according to claim 1, it is characterised in that the PBS of precooling used is dense It spends for 0.01M, PH is 7.2~7.4.
- 3. the isolation and culture method of gastric carcinoma cells according to claim 1, it is characterised in that step (3) described digestion Environment is 37 DEG C, 5%CO2Incubator, 0.1%~0.2% IV Collagenase Types digest 1~2h, then with 0.1%~0.2% 30~40min of Trypsin Induced.
- 4. the isolation and culture method of gastric carcinoma cells according to claim 3, it is characterised in that described 0.15% IV Collagenase Type digests 1.5h, then with containing 0.15% Trypsin Induced 35min.
- 5. the isolation and culture method of gastric carcinoma cells according to claim 4, it is characterised in that described 0.15% Trypsase contains 0.01% divinyl tetraacethyl disodium (EDTANa2)。
- 6. the isolation and culture method of gastric carcinoma cells according to claim 1, it is characterised in that the training described in step (4) It is dual anti-, 4~6 μ g/ml insulin containing 10% hyclone, penicillin containing 100u/ml and 100u/ml streptomysins to support base DMEM/F12 (1: 1) culture medium, pH 7.4.
- 7. the isolation and culture method of gastric carcinoma cells according to claim 1, it is characterised in that the training described in step (5) Support 3~4 μ g/cm of bottle2Poly-D-lysine coating, condition of culture be 37 DEG C, 5%CO2Incubator.
- 8. the isolation and culture method of gastric carcinoma cells according to claim 1, it is characterised in that thin described in step (6) Born of the same parents' purification process is to be purified with 0.25% Trypsin Induced.
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Cited By (3)
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| CN108865973A (en) * | 2018-06-20 | 2018-11-23 | 广西医科大学第附属医院 | The separation of SD Antrum interstitial cell and cultural method |
| CN110408595A (en) * | 2019-08-23 | 2019-11-05 | 厦门博创盛世生物技术有限公司 | A kind of cultural method of the tumour 3D organoid for gastric cancer test |
| CN114990052A (en) * | 2022-06-22 | 2022-09-02 | 中山大学附属第七医院(深圳) | Method for separating and extracting multiple subpopulations of extracellular vesicles derived from gastric cancer tissue |
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