CN104694460A - Culture medium for normal epithelial cell of human or mammal, culture methods, normal epithelial cell and application of normal epithelial cell - Google Patents
Culture medium for normal epithelial cell of human or mammal, culture methods, normal epithelial cell and application of normal epithelial cell Download PDFInfo
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Abstract
The invention relates to a culture medium for a normal epithelial cell of a human or mammal, primary separation culture, subculture, 3D gas-liquid culture and 3D matrigel culture methods, the normal epithelial cell generated by using the culture medium and the culture methods and application of the normal epithelial cell to a toxicological evaluation system. The culture medium is prepared by mixing DMEM and Ham's F-12NUTRIENT MIX according to the volume ratio of 3:1 and also adding 4-6% of fetal calf serum, 1-3nM triiodothyronine, 0.4-0.65% of insulin-transferrin-selenium reagent, 4-6mu g/ml transferrin, 9-11ng/mL epidermal growth factors, 0.3-0.5mu g/mL hydrocortisone, 0.5-1.5nM cholera toxin, 0.4-0.6mu g/mL amphoterrible B, 35-45mu g/mL gentamicin, 45-55nM calpeptin, 35-45ng/ml recombinant human IL-1RA and 3mu g/ml recombinant human R-Spondin-1. The culture medium disclosed by the invention can be used for carrying out separation culture or subculture on the normal epithelial cell of the human or the mammal and any other various tissue source, rapidly proliferating the normal epithelial cell in vitro and establishing a cell line; and the normal epithelial cell is a normal diploid cell and is applied to the toxicological evaluation system of the human or the mammal.
Description
Technical field
The present invention relates to cytobiology and toxicology field, be specifically related to the substratum of people or Mammals normal epithelium cell, primary separation and Culture, Secondary Culture, solution-air 3D cultivate and matrigel 3D cultural method, and above-mentioned substratum, normal epithelium cell that cultural method generates and the application in toxicological evaluation system thereof.
Background technology
Environmental pollution serious harm people's is healthy, and the respiratory system disease caused thus also constantly occurs.The epithelial cell of people stands in the breach the infringement target spot becoming main.The epithelial cell of organ specificity differentiation exercises important function, as the effect of the gaseous interchange of lung, the filtration of kidney, the removing toxic substances of liver and keying action, Islet Cells Insulin secretion effect and skin opposing hostile environment.Therefore, be used for probing into toxicant occupational exposure mechanism based on epithelial external model.In these researchs, use the cell strain of lung cancer cell line A549, Colon cancer cell line HT-29 and chromosomal variation, cause these to study the result obtained and produce notable difference and controversial each other.In order to avoid the disadvantageous effect that application cancer cells brings, scholars establish the method for various cellular immortalization, and the mode that they try to be transduceed by virus or oncogene in vitro establishes the clone of what is called " normally ".But genetic manipulation changes the genetic background of cell and real physiological status, can not reflect the truth function of normal epithelium cell, and the experimental measurements therefore obtained reduces greatly.
The epithelial cell of human organ specific differentiation is difficult to cultivate in vitro.From tissue sample, be directly separated the epithelial cell output obtained and cell purity very low, these are primary and the epithelial rate of propagation that goes down to posterity is also very limited, after the passage of limited number of time, propagation will be stopped, occur old and feeble and dead, this is with regard to the further application of restrictive cell culture technique.How obtaining the normal cell deriving from human body vitals, is a domestic and international Preclinic and clinic medical research difficult problem in the urgent need to address.Although establish a lot of cellular immortalization method at present, but major part is all, by the mode of virus importing, gene transfection, exogenous virus, proto-oncogene are imported object cell, its assortment of genes is random, its expression product can physiological pathway in interference cell, there is unexpected change in cell, compare with normal cell, its biological character such as cell caryogram, serum dependent form etc. may change, the characteristic even showing tumour cell had, the immortalized cells of foundation can not keep its original normal phenotype.But up to now, in toxicology, a kind of normal cell strain without channel genes immortalization also cannot be set up as research model.
Summary of the invention
The object of the invention is to the defect for overcoming prior art, and provide the substratum of a kind of people or Mammals normal epithelium cell, primary separation and Culture, Secondary Culture, solution-air 3D to cultivate and matrigel 3D cultural method, and above-mentioned substratum, normal epithelium cell that cultural method generates and the application in toxicological evaluation system thereof.
For achieving the above object, the present invention is by the following technical solutions: a kind of substratum for cultivator or mammiferous normal epithelium cell, described medium component comprises: the substratum of DMEM and Ham ' s F-12NUTRIENT MIX 3:1 mixing by volume, add 4-6% foetal calf serum simultaneously, 1-3nMtriiodothyronine, 0.4-0.65%insulin-transferrin-selenium reagent, 4-6 μ g/mltransferrin, 9-11ng/mL Urogastron, 0.3-0.5 μ g/mL hydrocortisone, 0.5-1.5nM Toxins,exo-, cholera, 0.4-0.6 μ g/mL amphotericin B, 35-45 μ g/mL gentamicin, 45-55nMcalpeptin, 35-45ng/ml recombinant human IL-1RA and 3ug/ml recombinant human R-Spondin-1.
A primary isolation cultivation method for people or mammiferous normal epithelium cell, comprises the steps:
The normal cell tissue sample of S1, collection clinical patients excision; Or collect mammiferous normal cell tissue sample;
S2, preparation Digestive system: described Digestive system is substratum described in the claim 1 containing collagenase/hyaluronic acid enzyme mixation;
S3, the normal cell tissue sample using ethanol to be separated with PBS buffer solution for cleaning successively, then normal cell tissue sample is put into the sterile petri dish containing precooling PBS damping fluid, under dissecting microscope, remove fat residual in normal cell tissue sample with dissecting tool;
S4, normal cell tissue sample put into containing step S2 prepare Digestive system centrifuge tube digest;
S5, postdigestive normal cell tissue sample low-speed centrifugal is removed supernatant;
S6, the normal cell tissue sample precipitation after step S5 process is resuspended in pancreatin/EDTA and digests;
The DMEM substratum that S7 and then row add containing FBS, low-speed centrifugal removes supernatant;
S8, add Dispase and DNase I, repeatedly blow and beat normal cell tissue sample with aseptic disposable plastic rifle head;
S9, again row add the DMEM containing FBS, use metre filter cell suspension, collect the cell suspension after filtering, low-speed centrifugal, remove supernatant;
Cell precipitation in S10, resuspended step S9, in substratum according to claim 1, is inoculated in culturing bottle and cultivates.
Concrete, the primary isolation cultivation method of people or mammiferous normal epithelium cell
The mixed solution of to be final concentration the be 1X of collagenase/Unidasa described in step S2;
The flesh tissue 1 time of separation washed by ethanol first with 95-100% in step S3, then uses PBS buffer solution for cleaning 2 times;
The condition digested in centrifuge tube in step S4 is 37 DEG C, duration 1-3 hour;
In step S5, low-speed centrifugal condition is low speed is 1000prm, and duration is 5 minutes;
In step S6, pancreatin/EDTA is 2-5ml, and concentration is 0.25%, is placed in resuspended under 1 hour or room temperature 10 minutes conditions on ice;
The low-speed centrifugal condition of step S7 is low speed is 1000prm, and duration is 5 minutes;
In step S8, Dispase is the 5mg/ml Dispase of 2ml temperature bath, and DNase I is the 1mg/mL DNaseI of 200 μ L, and piping and druming duration is 1 minute;
DMEM described in step S9 adds the DMEM of 10ml containing 10%FBS, and low-speed centrifugal condition is low speed is 1000prm, and duration is 5 minutes;
In step S10, culture condition is 37 DEG C, 5%CO
2.
A Secondary Culture method for people or mammiferous normal epithelium cell, comprises the steps:
S1, when people or mammiferous normal epithelium cell propagation to 70-90% abundance time, with 1xPBS buffer solution cell, then use pancreatin/EDTA to digest monolayer cell;
S2, use in DMEM substratum again and digestion reaction;
S3, low-speed centrifugal remove supernatant;
S4, to be deposited in described in claim 1 inoculation of medium with certain proportion re-suspended cell and to cultivate.
Concrete, the Secondary Culture method of people or mammiferous normal epithelium cell
In the cells frozen storing liquid that cell precipitation after step S3 process is resuspended in, be stored in liquid nitrogen for subsequent use.
Concrete, the Secondary Culture method of people or mammiferous normal epithelium cell
With 1xPBS buffer solution cell twice in step S2, described pancreatin/EDTA concentration 0.05%, digestion duration 2-5 minute;
In step S3, low-speed centrifugal condition is low speed is 1000prm, and duration is 5 minutes;
In step S4, selected ratio is 1:2,1:3,1:4 or 1:5, as long as maintain cell attachment and normal growth.
A solution-air 3D cultural method for people or mammiferous normal epithelium cell, comprises the steps:
Step S1, insert Tissue Culture Dish inner matter is put into culture dish;
The resuspended 2x10 of substratum described in step S2, use claim 1
5normal epithelium cell, is then inoculated in insert Tissue Culture Dish inner matter;
Suitable growth medium is added in step S3, culture dish;
Step S4, the culture dish being placed with inner matter is put into damp and hot incubator cultivate;
Step S5, substratum by inner matter in inner and peripheral culture dish are replaced by division culture medium;
Step S6, the culture dish being placed with inner matter is put into damp and hot incubator make to form compact siro spinning technology between cell in step S5;
Step S7, remove the inner and peripheral all substratum of inner matter, add division culture medium in peripheral culture dish, start 3D differentiation culture;
Step S8, in damp and hot incubator culturing cell 14 to 21 days, within every 2-3 days, change the substratum of inner matter periphery.
Concrete, the solution-air 3D cultural method of people or mammiferous normal epithelium cell
In step S4, culture condition is 37 DEG C, 5%CO
2,cultivating duration is 48 hours;
Cultivating duration in step S6 is 15-17 hour;
In step S8, culture condition is 37 DEG C, 5%CO
2.
A matrigel 3D cultural method for people or mammiferous normal epithelium cell, comprises the steps:
Step S1, by matrigel low temperature place nature melt;
Step S2, orifice surface paving skim matrigel in precooling, add surface and with rifle head uniform application by matrigel;
Step S3, by orifice plate place matrigel is solidified;
Step S4, digestion normal epithelium cell are extremely single and centrifugal;
Step S5, by resuspended for normal epithelium cell in matrigel;
Step S6, with rifle head, matrigel and normal epithelium cell to be mixed, mixture is joined and is covered with in advance in the orifice plate of matrigel, matrigel is solidified orifice plate placement to add substratum described in claim 1 again to add F substratum;
Step S7, described orifice plate is put in damp and hot incubator constant temperature culture.
Concrete, the matrigel 3D cultural method of people or mammiferous normal epithelium cell
In step S1, low temperature places nature thawing for spending the night thawing under 4 DEG C of conditions;
In step S3, placement condition is 37 DEG C, and duration is 15-30 minute;
In step S6, it is 37 DEG C that matrigel places curing condition, and duration is 30 minutes;
In step S7, culture condition is: 37 DEG C, 5%CO
2, constant temperature culture 10 days, changes 1 subculture in every two days.
The substratum of above-mentioned people or mammiferous normal epithelium cell, primary, go down to posterity, people that solution-air 3D or matrigel 3D cultural method are cultivated or mammiferous normal epithelium cell,
Described normal epithelium cell separation and Culture is from people or mammiferous dissimilar healthy tissues, and described normal epithelium cell does not import any foreign gene, is normal diploid cell, has the physiological function of normal differentiation.
Further, the normal epithelium cell of people or mammiferous different tissue sources, in toxicity detection and the application of setting up in toxicological evaluation system of environmental poisonous substance.
The present invention's beneficial effect is compared with prior art:
1. a kind of substratum provided by the invention, can be used for the normal epithelium cell that separation and Culture, Secondary Culture people or Mammals Various Tissues are originated.
2. a kind of normal epithelium cell separation and Culture provided by the invention, Secondary Culture method, can external fast breeding people or mammiferous normal epithelium cell set up clone.
3. people provided by the invention and mammiferous normal epithelium cell do not import any foreign gene, are normal diploid cell, had the physiological function of normal differentiation, can be applicable to people or mammiferous toxicological evaluation system by the display of 3D cultural method.
Below in conjunction with the drawings and specific embodiments, the invention will be further described.
Accompanying drawing explanation
Fig. 1 is the people of histological types and the cellular form of mouse normal epithelium cell;
Fig. 2 is that Human epithelium cells can normal differentiation under 3D culture condition;
Fig. 3 is that (A. karyotyping is for normal diploid for the experiment of people's normal bronchial epithelial cell (HNBEC) qualification normal physiological function; B. matrigel 3D cultivates as normal differentiation; It is normal that C.DNA damages answering; D. knurl is not become in transplanted tumor in nude mice experiment body);
Fig. 4 is that three kinds of environmental poisonous substances contrast the Morphology Effects of people's normal bronchial epithelial cell (HNBEC);
Fig. 5 is that three kinds of environmental poisonous substances contrast the impact of people's normal bronchial epithelial cell (HNBEC) survival rate;
Fig. 6 is that three kinds of environmental poisonous substances contrast the impact of people's normal bronchial epithelial cell (HNBEC) apoptosis.
Embodiment
In order to more fully understand technology contents of the present invention, below in conjunction with specific embodiment technical scheme of the present invention being introduced further and illustrating.
Embodiment 1 for separating of cultivating, the substratum of Secondary Culture people or mammiferous normal epithelium cell
Its moiety comprises: the mixed culture medium of DMEM and Ham ' s F-12NUTRIENT MIX (GIBCO), volume proportion is 3:1, add 5% foetal calf serum (GIBCO) simultaneously, 2nM triiodothyronine (Sigma), 0.5%insulin-transferrin-selenium reagent (Life Technologies), 5 μ g/mltransferrin (Life Technologies), 10ng/mL Urogastron (Sigma), 0.4 μ g/mL hydrocortisone (Sigma), 1nM Toxins,exo-, cholera (List Biological Labs, Campbell, CA), 0.5 μ g/mL amphotericin B (Fungizone, Bristol-Myers Squibb, Park Avenue NY), 40 μ g/mL gentamicin (Gentacin, Schering-Plough, Keni lworth, NJ), 50nM calpeptin (ENZO LifeSciences, Farmingdale, NY), 40ng/ml recombinant human IL-1RA (PeProTech), 3ug/ml recombinant human R-Spondin-1 (PeProTech).
The primary isolation cultivation method of embodiment 2 people or Mammals normal epithelium cell
1) when patient or patient care people informed consent, the normal tissue sample of clinical patients excision is collected; Or strictly follow experimentation on animals operating process and the regulations of ARRIVE (Animal Research:Reporting of In Vivo Experiments), collect mammiferous normal tissue sample;
2) preparation of Digestive system: (composition is the mixed culture medium of DMEM and Ham ' s F-12NUTRIENT MIX (GIBCO) containing the above-mentioned substratum of collagenase/Unidasa (final concentration is the mixed solution of 1x), volume proportion is 3:1, add 5% foetal calf serum (GIBCO) simultaneously, 2nM triiodothyronine (Sigma), 0.5%insulin-transferrin-selenium reagent (Life Technologies), 5 μ g/ml transferrin (LifeTechnologies), 10ng/mL Urogastron (Sigma), 0.4 μ g/mL hydrocortisone (Sigma), 1nM Toxins,exo-, cholera (List Biological Labs, Campbell, CA), 0.5 μ g/mL amphotericin B (Fungizone, Bristol-Myers Squibb, Park Avenue NY), 40 μ g/mL gentamicin (Gentacin, Schering-Plough, Kenilworth, NJ), 50nM calpeptin (ENZO Life Sciences, Farmingdale, NY), 40ng/ml recombinant human IL-1RA (PeProTech), 3ug/ml recombinant human R-Spondin-1 (PeProTech).Determine the consumption of Digestive system according to the size of flesh tissue, usually need 10 times to the consumption of the Digestive system of sample volume
3) wash the flesh tissue 1 time of separation with the ethanol of 95-100%, then wash 2 times with PBS, then by organizing the sterile petri dish put into containing precooling PBS, under dissecting microscope, with dissecting tweezers and fat residual in tissue removed by scissors.
4) tissue sample is put into 14ml or the 50ml centrifuge tube containing above-mentioned Digestive system, 37 DEG C of digestion 1-3 hour.
5) organize low-speed centrifugal (1000rpm) 5 minutes by postdigestive, remove supernatant.
6) cell precipitation is resuspended in the 0.25% pancreatin/EDTA of 2-5mL, is placed in 1 hour or room temperature 10 minutes on ice.
7) then add the DMEM substratum of 10ml containing 10%FBS, centrifugal 5 minutes of low speed 1000rmp, supernatant is removed clean as far as possible.
8) add the 5mg/mL Dispase of 2ml temperature bath and the 1mg/mL DNase I of 200 μ L, repeatedly blow and beat sample 1 minute with aseptic P1000 disposable plastic rifle head.
9) add the DMEM of 10ml containing 10%FBS, with the metre filter cell suspension in 40-70 μm of aperture, collect the cell suspension after filtering, centrifugal 5 minutes of low speed 1000rmp, remove supernatant.
10) re-suspended cell is deposited in above-mentioned substratum, and the culturing bottle being inoculated in T25 or T75 is cultivated, and culture condition is 37 DEG C, 5%CO2.
The Secondary Culture method of embodiment 3 people or Mammals normal epithelium cell
1) when people or mammiferous normal epithelium cell propagation are to 70-90% abundance, with 1xPBS washed cell twice, 0.05% pancreatin/EDTA digests monolayer cell 2-5 minute.
2) 10ml is completely in DMEM and digestion reaction.
3) 1000rmp removes supernatant in centrifugal 5 minutes.
4) with certain proportion, as 1:2:3,1:4,1:5, as long as cell attachment and normal growth can be maintained) re-suspended cell is deposited in culture medium inoculated in 10ml embodiment 1 and cultivates.
5) if desired can by 1x10
6epithelial cell is resuspended in the cells frozen storing liquid (90% foetal calf serum and 10%DMSO) of 1-2ml, is stored in liquid nitrogen for subsequent use.
The normal epithelium cell of the different tissue sources of separation and Culture, the successful people of Secondary Culture and mouse according to the method described above, the form of basis of microscopic observation cell is as Fig. 1.
The solution-air 3D of embodiment 4 people or Mammals normal epithelium cell cultivates
1) the Millicell PCF inner matter (12mm size, Millipore) of 0.4 μm is put into the culture dish of 60mm.
2) with the resuspended 2x10 of substratum in the embodiment 1 of 400 μ l
5cell, is then inoculated in each inner matter.
3) the suitable growth medium of 11ml is added in each culture dish.
4) culture dish being placed with inner matter is put into 37 DEG C, 5%CO
2cultivate 48 hours in damp and hot incubator.
5) substratum in inner matter inside and peripheral culture dish is replaced by division culture medium (CELLnTEC, Switzerland)
6) culture dish being placed with inner matter is put into damp and hot incubator to make to form compact siro spinning technology between cell for about 16 hours.
7) remove the substratum that inner matter is inner and periphery is all, in peripheral culture dish, add the division culture medium of 3.2ml, start 3D differentiation culture.
8) at 37 DEG C, 5%CO
2culturing cell 14 to 21 days in damp and hot incubator.Within every 2-3 days, change the substratum of inner matter periphery.
The matrigel 3D of embodiment 5 people or Mammals normal epithelium cell cultivates
1) to spend the night under matrigel (BD, BD Biosciences) being placed the condition of 4 DEG C thawing.
2) at 6 orifice surface paving skim matrigels of precooling, the matrigel of 120ul added surface and use rifle head uniform application.
3) 6 orifice plates are placed 37 DEG C of about 15-30 minute, matrigel is solidified.
4) normal epithelium cell is digested extremely single and centrifugal.
5) gently by resuspended for cell in the matrigel of 1.2ml.
6) being joined by mixture after matrigel and cell being mixed gently with rifle head is covered with in 6 orifice plates of matrigel in advance, and suggestion cell count is 1x10
5/ hole.6 orifice plates are placed under 37 DEG C of conditions and matrigel was solidified in about 30 minutes.Add the substratum in 1ml embodiment 1 again.
Above-mentioned 6 orifice plates are positioned over 37 DEG C, the damp and hot incubator constant temperature culture of 5%,CO2 10 days, within every 2 days, change 1 subculture.
The normal epithelium cell of the people cultivated according to above-mentioned 3D cultural method or the different tissue sources of mouse, basis of microscopic observation after section, dyeing.As Fig. 2, people's normal bronchial epithelial cell normal differentiation formation can have ciliary structures, people's normal mammary epithelial and prostate epithelial cell normal differentiation expression specificity differentiation marker thing.
According to the substratum of above-mentioned people or mammiferous normal epithelium cell, primary, go down to posterity, people that solution-air 3D or matrigel 3D cultural method are cultivated or mammiferous normal epithelium cell, these normal epithelium cell separation and Culture are from people or mammiferous dissimilar healthy tissues, normal epithelium cell does not import any foreign gene, for normal diploid cell, there is the physiological function of normal differentiation, as shown in Figure 3.
Embodiment 6 people or Mammals normal epithelium cell are applied to the toxicity detection (cell proliferation) of environmental poisonous substance
1) epithelial cell is inoculated in 96 porocyte culture plates, treats that cytogamy degree reaches about 80%, contaminate.
2) Nano-meter SiO_2
2: by Nano-meter SiO_2 before contamination
2the ultrasonic 0.5h of Ultrasonic Cell Disruptor, is then placed on uv irradiating 0.5h in Biohazard Safety Equipment.With serum free medium by Nano-meter SiO_2
2be configured to following concentration gradient 40 μ g/ml, 80 μ g/ml, 120 μ g/ml, 160 μ g/ml, 200 μ g/ml, add in Tissue Culture Plate successively, and the multiple hole of each gradient 6, is placed in cell culture incubator and cultivates 24h.
Sexavalent chrome: with serum free medium, sexavalent chrome is configured to following concentration gradient 5 μMs, 10 μMs, 15 μMs, 20 μMs, 30 μMs, adds successively in Tissue Culture Plate, be placed in cell culture incubator and cultivate 24h.
Benzopyrene: with serum free medium, benzopyrene is configured to following concentration gradient 20 μMs, 40 μMs, 80 μMs, 100 μMs, 120 μMs, adds successively in Tissue Culture Plate, be placed in cell culture incubator and cultivate 24h.
3) after 24h, observation of cell morphological change, and under inverted phase contrast microscope Taking Pictures recording.
4) discarded by the substratum in culture plate, every hole adds the CCK-8 reagent mixing of 90 μ l substratum and 10 μ l, is placed in cell culture incubator by culture plate and hatches 1-4h.
5) be determined at the absorbance at 450nm wavelength place by microplate reader, 630nm is as reference wavelength.Measure the survival activity of cell.According to above-mentioned environmental poisonous substance to the epithelial detection method of toxicity of people's normal bronchial, observation of cell form (Fig. 4) after contamination 24h, with the tracheal epithelial cell 16HBE of channel genes immortalization in contrast, people's normal bronchial epithelial cell, after nanosized SiO_2, sexavalent chrome Cr (VI), benzopyrene B (a) P tri-kinds of toxicant contaminations, morphological change is all more obvious.Measure cells survival activity (Fig. 5) through microplate reader, although three kinds of poisonous substances all cause two kinds of cytoactives greatly to reduce, people's normal bronchial epithelial cell compares 16HBE cell, all more responsive to these three kinds of toxicants.
Embodiment 7 people or Mammals normal epithelium cell are applied to the toxicity detection (apoptosis) of environmental poisonous substance
1) epithelial cell is inoculated in 6 porocyte culture plates, when cytogamy degree reaches about 80%, carries out cell nano SiO
2contamination, the same embodiment 6 of method, Nano-meter SiO_2
2concentration gradient 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 30 μ g/ml.
2) contaminate after 24h, discarded by substratum, PBS washes twice, with the trypsin digestion cell containing EDTA, collecting cell in 1.5ml centrifuge tube,
3) the centrifugal 5min of 2000rpm, abandons supernatant, adds PBS re-suspended cell,
4) the centrifugal 5min of 2000rpm, abandons supernatant, comes again.
5) add the 1x binding buffer liquid re-suspended cell of 500 μ l, in cell, add 5 μ l Annexin V-FITC successively, 5 μ l PI, mixing
6) under room temperature, lucifuge hatches 15min, detects at flow cytometer.Interpretation of result illustrates carry out according to Annexin V, FITC apoptosis detection kit (Japanese Dojindo company).
According to above-mentioned environmental poisonous substance to the epithelial detection method of toxicity of people's normal bronchial, after contamination 24h, detect apoptosis (Fig. 6).Nanosized SiO_2 can cause two kinds of apoptosis, and becomes dependency relationships with poisoning dosage.But people's normal bronchial epithelial cell compares 16HBE cell, apoptosis occurs more obvious, also more responsive.
The above only further illustrates technology contents of the present invention with embodiment, so that reader is easier to understand, but does not represent embodiments of the present invention and is only limitted to this, and any technology done according to the present invention extends or recreation, all by protection of the present invention.
Claims (12)
1. the substratum for cultivator or mammiferous normal epithelium cell, it is characterized in that, described medium component comprises: the substratum of DMEM and Ham ' s F-12NUTRIENT MIX 3:1 mixing by volume, add 4-6% foetal calf serum simultaneously, 1-3nM triiodothyronine, 0.4-0.65%insulin-transferrin-selenium reagent, 4-6 μ g/ml transferrin, 9-11ng/mL Urogastron, 0.3-0.5 μ g/mL hydrocortisone, 0.5-1.5nM Toxins,exo-, cholera, 0.4-0.6 μ g/mL amphotericin B, 35-45 μ g/mL gentamicin, 45-55nM calpeptin, 35-45ng/ml recombinant human IL-1RA and 3ug/ml recombinant human R-Spondin-1.
2. a primary isolation cultivation method for people or mammiferous normal epithelium cell, is characterized in that, comprise the steps:
The normal cell tissue sample of S1, collection clinical patients excision; Or collect mammiferous normal cell tissue sample;
S2, preparation Digestive system: described Digestive system is substratum described in the claim 1 containing collagenase/hyaluronic acid enzyme mixation;
S3, the normal cell tissue sample using ethanol to be separated with PBS buffer solution for cleaning successively, then normal cell tissue sample is put into the sterile petri dish containing precooling PBS damping fluid, under dissecting microscope, remove fat residual in normal cell tissue sample with dissecting tool;
S4, normal cell tissue sample put into containing step S2 prepare Digestive system centrifuge tube digest;
S5, postdigestive normal cell tissue sample low-speed centrifugal is removed supernatant;
S6, the normal cell tissue sample precipitation after step S5 process is resuspended in pancreatin/EDTA and digests;
The DMEM substratum that S7 and then row add containing FBS, low-speed centrifugal removes supernatant;
S8, add Dispase and DNase I, repeatedly blow and beat normal cell tissue sample with aseptic disposable plastic rifle head;
S9, again row add the DMEM containing FBS, use metre filter cell suspension, collect the cell suspension after filtering, low-speed centrifugal, remove supernatant;
Cell precipitation in S10, resuspended step S9, in substratum according to claim 1, is inoculated in culturing bottle and cultivates.
3. the primary isolation cultivation method of people according to claim 2 or mammiferous normal epithelium cell, is characterized in that,
The mixed solution of to be final concentration the be 1X of collagenase/Unidasa described in step S2;
The flesh tissue 1 time of separation washed by ethanol first with 95-100% in step S3, then uses PBS buffer solution for cleaning 2 times;
The condition digested in centrifuge tube in step S4 is 37 DEG C, duration 1-3 hour;
In step S5, low-speed centrifugal condition is low speed is 1000prm, and duration is 5 minutes;
In step S6, pancreatin/EDTA is 2-5ml, and concentration is 0.25%, is placed in resuspended under 1 hour or room temperature 10 minutes conditions on ice;
The low-speed centrifugal condition of step S7 is low speed is 1000prm, and duration is 5 minutes;
In step S8, Dispase is the 5mg/ml Dispase of 2ml temperature bath, and DNase I is the 1mg/mL DNase I of 200 μ L, and piping and druming duration is 1 minute;
DMEM described in step S9 adds the DMEM of 10ml containing 10%FBS, and low-speed centrifugal condition is low speed is 1000prm, and duration is 5 minutes;
In step S10, culture condition is 37 DEG C, 5%CO
2.
4. a Secondary Culture method for people or mammiferous normal epithelium cell, is characterized in that, comprise the steps:
S1, when people or mammiferous normal epithelium cell propagation to 70-90% abundance time, with 1xPBS buffer solution cell, then use pancreatin/EDTA to digest monolayer cell;
S2, use in DMEM substratum again and digestion reaction;
S3, low-speed centrifugal remove supernatant;
S4, to be deposited in described in claim 1 inoculation of medium with certain proportion re-suspended cell and to cultivate.
5. the Secondary Culture method of people or mammiferous normal epithelium cell according to claim 4, is characterized in that, in the cells frozen storing liquid that the cell precipitation after step S3 process is resuspended in, be stored in liquid nitrogen for subsequent use.
6. the Secondary Culture method of people or mammiferous normal epithelium cell according to claim 4, is characterized in that,
With 1xPBS buffer solution cell twice in step S2, described pancreatin/EDTA concentration 0.05%, digestion duration 2-5 minute;
In step S3, low-speed centrifugal condition is low speed is 1000prm, and duration is 5 minutes;
In step S4, selected ratio is 1:2,1:3,1:4 or 1:5, as long as maintain cell attachment and normal growth.
7. a solution-air 3D cultural method for people or mammiferous normal epithelium cell, is characterized in that, comprise the steps:
Step S1, insert Tissue Culture Dish inner matter is put into culture dish;
The resuspended 2x10 of substratum described in step S2, use claim 1
5normal epithelium cell, is then inoculated in insert Tissue Culture Dish inner matter;
Suitable growth medium is added in step S3, culture dish;
Step S4, the culture dish being placed with inner matter is put into damp and hot incubator cultivate;
Step S5, substratum by inner matter in inner and peripheral culture dish are replaced by division culture medium;
Step S6, the culture dish being placed with inner matter is put into damp and hot incubator make to form compact siro spinning technology between cell in step S5;
Step S7, remove the inner and peripheral all substratum of inner matter, add division culture medium in peripheral culture dish, start 3D differentiation culture;
Step S8, in damp and hot incubator culturing cell 14 to 21 days, within every 2-3 days, change the substratum of inner matter periphery.
8. the solution-air 3D cultural method of people or mammiferous normal epithelium cell according to claim 7, is characterized in that,
In step S4, culture condition is 37 DEG C, 5%CO
2, cultivating duration is 48 hours;
Cultivating duration in step S6 is 15-17 hour;
In step S8, culture condition is 37 DEG C, 5%CO
2.
9. a matrigel 3D cultural method for people or mammiferous normal epithelium cell, is characterized in that, comprise the steps:
Step S1, by matrigel low temperature place nature melt;
Step S2, orifice surface paving skim matrigel in precooling, add surface and with rifle head uniform application by matrigel;
Step S3, by orifice plate place matrigel is solidified;
Step S4, digestion normal epithelium cell are extremely single and centrifugal;
Step S5, by resuspended for normal epithelium cell in matrigel;
Matrigel and normal epithelium cell mix by step S6, use rifle head, are joined by mixture and are covered with in advance in the orifice plate of matrigel, matrigel is solidified orifice plate placement and add substratum according to claim 1 again;
Step S7, described orifice plate is put in damp and hot incubator constant temperature culture.
10. the matrigel 3D cultural method of people or mammiferous normal epithelium cell according to claim 9, is characterized in that,
In step S1, low temperature places nature thawing for spending the night thawing under 4 DEG C of conditions;
In step S3, placement condition is 37 DEG C, and duration is 15-30 minute;
In step S6, it is 37 DEG C that matrigel places curing condition, and duration is 30 minutes;
In step S7, culture condition is: 37 DEG C, 5%CO
2, constant temperature culture 10 days, changes 1 subculture in every two days.
11. according to the substratum of the arbitrary described people of claim 1 to 10 or mammiferous normal epithelium cell, primary, go down to posterity, people that solution-air 3D or matrigel 3D cultural method are cultivated or mammiferous normal epithelium cell, it is characterized in that,
Described normal epithelium cell separation and Culture is from people or mammiferous dissimilar healthy tissues, and described normal epithelium cell does not import any foreign gene, is normal diploid cell, has the physiological function of normal differentiation.
The normal epithelium cell of 12. people according to claim 11 or mammiferous different tissue sources, in toxicity detection and the application of setting up in toxicological evaluation system of environmental poisonous substance.
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