CN106086153A - Caco 2 cell based on 2D and the 3D evaluation methodology to nano zine oxide biological safety - Google Patents
Caco 2 cell based on 2D and the 3D evaluation methodology to nano zine oxide biological safety Download PDFInfo
- Publication number
- CN106086153A CN106086153A CN201610477781.2A CN201610477781A CN106086153A CN 106086153 A CN106086153 A CN 106086153A CN 201610477781 A CN201610477781 A CN 201610477781A CN 106086153 A CN106086153 A CN 106086153A
- Authority
- CN
- China
- Prior art keywords
- cell
- culture
- nano
- nano zine
- zine oxide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Caco 2 cell based on 2D and the 3D evaluation methodology to nano zine oxide biological safety, belongs to mensuration or the method for inspection technical field of microorganism, comprises the steps: 1) cultivation of 2D cell；2) 3D cell is cultivated；3) passage；4) nano-zinc oxide powder is dispersed in the DMEM culture medium of serum-free standby；5) carry out Characterization of Nano-materials and morphological observation, use MTT colorimetric determination cytoactive, inflammatory factor interleukin 8 is detected, by the cell death way that flow cytomery is different；Use SPSS19.0 statistical software to be analyzed, data are carried out one factor analysis of variance, calculate the significance of difference of p < 0.05 and p < 0.01.The present invention have evaluated in 2D and 3D cell culture model nano zine oxide to the biological effect of cell and to nano oxidized zinc toxicity.These researchs have certain theory significance for the intestinal toxicity of Study in vitro nano zine oxide, and the formulation for nano zine oxide limit standard provides reference.
Description
Technical field
The invention belongs to mensuration or the method for inspection technical field of microorganism, be particularly related to Caco-2 based on 2D and 3D
The cell evaluation methodology to nano zine oxide biological safety.
Background technology
At present, the research of environment and Human health effects is gone back imperfection by nano material, and relevant information is fewer.With often
Rule food material is compared, and nanometer food material particle size is less, differs greatly physics and chemistry is qualitative with it, and nano material exists
Biological activity in body, and the biological positive seondary effect produced all is amplified, the cytotoxicity of induction is likely to strengthen.
Accordingly, it would be desirable to toxicology authority's information of a large amount of science, it is dangerous in explaining what dosage range, in what dosage range is
Safety.
In numerous metal nano materials, nano zine oxide is one of material of being widely used.Nano zine oxide is
A kind of particle diameter 1～100nm novel high function fine inorganic material.Compared with regular oxidation zinc, nano zine oxide has
The uviolresistance of extremely strong non-oxidizability, corrosion resistance, photocatalytic and uniqueness so that it is at photoelectricity, rubber industry, coating
The aspects such as pottery, cosmetics, food additive, biomedicine are widely used, and will in fields such as light, sound, electricity nano-devices
Have broad application prospects.
It is currently known nano zine oxide and can pass through the approach entrance machines such as contact skin, respiratory tract, digestive tract and intravenous injection
Body, but body is difficult to get rid of this kind of small material, the most potential certain poisonous effect.Material by after nanorize, its toxicity, suction
Receive and discharge regime all changes.The cell toxicology research of nano zine oxide is carried out relatively broad, it has therefore proved that nano oxidized
The various kinds of cell systems such as human lung adenocarcinoma cell, human colon cancer cell, human embryonic lung fibroblast and airway epithelial cell are had by zinc
Toxic action.
Cell is the ultimate unit of most of organism, and its change generally can reflect the change of living organism function and structure
Change, and when body function does not changes with structure, cell function and structure are it can also happen that change.Cell toxicology is main
Be physics in application cell in vitro technological means research environment, the multiple pollutant such as chemical and biological to the damage effect of cell with
Rule, thus the cellulotoxic effect that obtains this material exposed features relevant with dosage is (such as open-assembly time, route of exposure, exposure
Frequency), all multi information such as toxic action mechanism.Carry out nano-particle biological effect research with cell for object of study, mainly should
With the cell strain of In vitro culture, nano-particle is carried out toxic effect monitoring, evaluate nano-particle harm issuable to human body.
General test method is that passage cell or primary cell are placed in plastic culture dish, adds a certain amount of nanometer material
Material, then observation of cell reaction.The cell type currently mainly used includes phagocyte, neurocyte, epithelial cell, endothelium
Cell, erythrocyte and various cancerous cell.The project that predominantly detects related at present include cytotoxicity detection, apoptosis detection with
And immunocytochemical technique.
Tracing back to eighties of last century the seventies, various countries' scientists recognizes that the external environment of growth of cancer cells is to its phenotype shape
One-tenth is very important.A lot of researchs show that normal tissue cell and cancerous cell are trained in conventional two-dimensional (2D) plastic culture dish
Foster meeting loses its original organizational structure and performance, compares cells in vivo and often carries out changing of metabolism, phenotype and gene expression
Becoming, these change development and the function influencing whether cancerous cell.Brinster et al. finds to be inoculated into little by embryo tumor cell
Define embryonal carcinoma under Corium Mus, secondary cell is inoculated in the most pregnant Development of Mouse Embryos foetal sac and then can develop into mice, this result
Show that the environment variations of growth of tumour cell is the biggest on the probability impact of its formation tumor.Set up in these theoretical basis
On, Emerman, Elsdale and Weaver et al. propose to utilize to reproduce extracellular matrix (extracellular
Matrix, ECM) to constitute grid structure support and connection organizational structure carrys out the outer 3D cell culture model of construct.
3D cell is cultivated and is defined as cell, some somatomedin and reproduces stromatin and skeleton co-cultures, to imitate
The characteristic of cells in vivo growing environment.By imitating the characteristic of internal milieu, and utilize traditional cell culture studies instrument, 3D
Cell is cultivated and is provided the visual angle of uniqueness to observe the evolution of the behavior of stem cell, histoorgan and tumor.
Cell, in the superficial growth of plastic culture dish, i.e. can only grow in two dimension (2D) plane, but in vivo, cell is then
By other cells, tissue and extracellular matrix tight encirclement.Scientists was thin to external 2D from molecule and gene level in the past
Born of the same parents study, to explore the impact on cell behaviors of specific molecular and gene alteration.Although internal animal model is more
The metabolism of cell, phenotype and gene expression can be reflected exactly, but the research realizing large-scale molecular level is also extremely difficult
's.Set up external 3D culture model and will assist in the wide gap crossed between the cultivation of 2D cell and animal experiment, because the training of 3D cell
Support traditional 2D cell culture studies instrument that can utilize, and interior state offer cell growth support is provided, combine 2D thin
Born of the same parents cultivate and the advantage of animal model.3D cell is cultivated can be with the microenvironment of analog cell growth, analogue body inner cell and cell
The interaction of substrate, accurately reflects the mechanism of effect between cancerous cell and interstitial.Meanwhile, the cell that 3D cell model is built
Microenvironment is also more suited for inducing the polarity of cell, and this is critically important to the direct secretion of tissue and its product.
Along with people contact with nano zine oxide increasing, the genotoxic potential of human health is affected and also result in by it
Pay high attention to.Research nano material in Cytotoxic numerous tests, two dimension (2D) monolayer cell culture remain account for leading
The external model of status.The cell cultivated in tradition 2D plastic culture dish can lose its original organizational structure, polarity and
Protein protein interaction, and compare In vivo model and often change the metabolism of cell, phenotype and gene expression.
When two dimension cell culture model expands to nanotoxicology research, it these limit be likely to result in obtaining misleading result and
The risk of conclusion.All cells the most in vivo is all grown in three-dimensional (3D) environment, the phenotype of individual cells and
Function is to be highly dependent on the complicated interaction of three-dimensional tissue-extracellular matrix (ECM) albumen and adjacent cells.
Therefore, the toxicity research carrying out nanoparticle in the external 3D model of an altitude simulation body physiological environment can
Think that the Evaluation of Biocompatibility of nano material provides more theoretical basiss and reference value.
Summary of the invention
It is an object of the invention to overcome defect mentioned above and deficiency, and Caco-2 cell based on 2D and 3D is provided
Evaluation methodology to nano zine oxide biological safety.
The technical scheme that the present invention realizes the employing of its purpose is as follows.
The Caco-2 cell based on 2D and the 3D evaluation methodology to nano zine oxide biological safety, it is characterised in that bag
Include following steps:
1) 2D cell is cultivated: add Caco-2 cell strain containing 10% FBS and the complete DMEM culture medium of 1% L-glutaminate
In, 37 DEG C, 5% CO2 quiescent culture, a cell culture fluid was changed every 2 ~ 3 days, trophophase cell of taking the logarithm carries out follow-up examination
Test；
2) 3D cell is cultivated: is placed in by the Matrigel glue of sterile centrifugation tube subpackage before the test and dissolves 2 ~ 3 h on ice, then will move
Liquid device rifle head and 96 well culture plates are placed in cooled on ice 1 h；Take 10 ~ 20 μ L Matrigel glue uniform embedding cell culture plates
Bottom, is placed in 37 DEG C of cell culture incubator 30 min and makes it solidify；Digestion two dimension cell, counts and is diluted to 2 x 104/ mL, point
Do not draw 50 μ L cell suspension and Matrigel glue, proceed to 96 well culture plates after mixing by 1:1, then be placed in 37 DEG C of cells cultivations
Case 30 min makes it solidify；Again the glue upper strata solidified drips 100 μ L and contains the complete of 10% FBS and 1% L-glutaminate
DMEM culture medium, is 37 DEG C, 5% CO in condition2Incubator in quiescent culture, changed once completely DMEM every 2 ~ 3 days and cultivate
Base；
3) passage: Caco-2 cell attachment grows, and through 3-4 d, when cell grows to monolayer 80%-95%, discards supernatant
Liquid, rinses gently with appropriate PBS, discards waste liquid, adds 0.25% trypsin and carries out digesting 3-5 min, under inverted microscope
Observing when cell just becomes round, the DMEM culture medium adding a small amount of 10% hyclone terminates cell dissociation process, inhales with suction pipe
Taking culture medium, piping and druming makes cell the most adherent gently, is transferred in new culture bottle by cell suspension, and adds appropriate cultivation
Base, the condition of putting into is 37 DEG C, 5% CO2Incubator in cultivate after 3-4 d, carry out Secondary Culture next time；
4) nano-zinc oxide powder is dispersed in the DMEM culture medium of serum-free, makes variable concentrations nano zine oxide suspension；
Put in 4 DEG C of refrigerators standby；Before using, ultrasonic cell disruption instrument need to be used to make nano material depolymerization, ultrasonic bar every time
Part is: 3 min, surpasses 3 s and stops 2 s, power 300 w；
5) carry out Characterization of Nano-materials and morphological observation, use MTT colorimetric determination cytoactive, white to inflammatory factor
Cytokine-8(IL-8) detect, by the 2D cell death form after the double dye of fluorescence microscope AO/EB, by stream
The cell death way that the detection of formula cell instrument is different；Use SPSS19.0 statistical software to be analyzed, data are carried out single factor test
Variance analysis, calculates the significance of difference of p < 0.05 and p < 0.01.
Further, in step 5), described Characterization of Nano-materials, comprise the following steps: nano-particle is ultrasonic by dehydrated alcohol
Suspendible, is added drop-wise on copper mesh be dried microscopy under transmission electron microscope；Use Malvern particle instrument that nano-particle is carried out intensity and particle diameter
The analysis of distribution.
Further, in step 5), described morphological observation, comprise the following steps: by under 2D and 3D condition of culture
Caco-2 cell, carries out contamination process, and cell is the 24 nm nano zine oxide contamination solution of 10 and 75 μ g/mL respectively with concentration
Co-culture 12 h；After hatching 12 h, observation of cell form under inverted microscope, and shoot photo；Cellular control unit is and nothing
The DMEM culture medium of serum co-cultures.
Further, in step 5), use MTT colorimetric determination cytoactive, comprise the following steps: 2D and 3D cell is entered
Row contamination processes, and i.e. adds tri-kinds of particle diameters of 24,56,87 nm of 0,10,25,50,75,100 μ g/ml variable concentrations
Nano zine oxide contamination liquid and co-culture of cells 12 h；After contamination time terminates, PBS rinse 2 times, every hole adds 0.05 mg/mL
MTT200 μ L, carry out after 4 h hatch, absorbing MTT supernatant, adding 150 μ L DMSO on shaking table, carry out low speed concussion
15 min, the purple crystal thing of intracellular formation fully dissolves；The OD value at 570 nm is measured by multi-functional microplate reader；In test
If zeroing group: DMEM, MTT and DMSO；Matched group: cell, DMEM, MTT and DMSO；3D cultivates needs and sets without cell, containing only
There is the blank group of gel；
Calculating cell viability, formula is as follows:
Cell viability=OD (Aexp)-OD (Aneg)/OD (Acon)-OD (Aneg)
Wherein, Aexp is test group；Aneg is zeroing group；Acon is test control group.
Further, in step 5), cross the 2D cell death form after the double dye of fluorescence microscope AO/EB, including following
Step:
Select to be in the Caco-2 cell of exponential phase, use 0.25% trypsinization, add appropriate DMEM immediately complete
Full culture medium, the cell suspension utilizing cell counting count board furnishing density to be 2 x 105/mL, it is inoculated in 6 well culture plates and cultivates 12 h
After, then co-culture 24 h with tri-kinds of grain diameter nano zinc oxide contamination solution of 24,56 and 87 nm that concentration is 10,75 μ g/mL；
After hatching 24 h, PBS rinse 2 times, add 300 μ L molten containing the PBS of 100 ug/mL acridine oranges and 100 ug/mL ethidium bromides
Liquid, with PBS rinse 2 times after dyeing 3min, at fluorescence microscopy Microscopic observation coloration result, and preservation of taking pictures；Cellular control unit is
Co-culture with the DMEM culture medium of serum-free.
Further, in step 5), inflammatory factor interleukin 8 (IL-8) is detected, comprises the following steps:
(1) reagent is prepared:
Use IL-8 detection kit that IL-8 protein level is detected；The standard of reagent is carried out according to test kit description
Standby, draw standard curve；
(2) sample is prepared:
2D and 3D cell is carried out contamination process, i.e. adds three kinds of grain diameter nano zinc oxide of 0 ~ 100 μ g/mL variable concentrations
Contamination liquid and co-culture of cells 12 h；After contamination time terminates, 1000 rpm, centrifugal 10 min are to remove some polymer and to receive
Rice grain, collects supernatant and proceeds to 96 new orifice plates, pending follow-up test；
(3) sample is analyzed:
Microplate reader measures the OD value in each hole at 450 nm wavelength, calculates sample concentration.
Further, in step 5), by the cell death way that flow cytomery is different, use Annexin V-
Alexa Fluor 488 method, comprises the following steps:
Under 2D condition of culture, carry out cell contamination process, i.e. add 24 nm nano oxygen of 0 ~ 100 μ g/mL variable concentrations
Change zinc contamination liquid and co-culture of cells 24 h；After contamination time terminates, collect floating cells and abandon upper strata nano zine oxide contamination
Liquid, PBS 2 times, cell, pending follow-up test are collected in digestion；
Under 3D condition of culture, carry out cell contamination process, i.e. add 24 nm nano zine oxide contamination liquid of variable concentrations with thin
Born of the same parents co-culture 24 h.After contamination time terminates, abandon upper strata nano zine oxide contamination liquid, add the 3D cell recovered liquid of 100 L,
Being swept by glue-line gently, is placed in and melts 30 min on ice, shakes up once every 10 min, treats that cell precipitation can sink to smoothly
At the bottom of pipe, illustrate that Matrigel gum base originally melts completely；Now, 2000 rpm, centrifugal 5 min, abandon supernatant, pending follow-up examination
Test；
The above-mentioned every hole of 2D and 3D cell sample is added the 1X Annexin-binding buffer re-suspended cell of 100 μ L.To
Every hole adds the 100 μ g/mL PI working solutions of 5 μ L Annexin V-Alexa Fluor 488 and 1 μ L respectively, at room temperature
Flow cytometer is utilized to detect fluorescent value at 530 nm and 575 nm after hatching 15 min immediately.
The evaluation methodology to nano zine oxide biological safety of the Caco-2 cell based on 2D and 3D of the present invention, have evaluated
In different cell culture models, nano zine oxide is to the biological effect of cell and to nano oxidized zinc toxicity.These researchs
Intestinal toxicity for Study in vitro nano zine oxide has certain theory significance, and is the formulation of nano zine oxide limit standard
Thering is provided reference, the safety problem for research nano zine oxide further provides reliable scientific basis, for other nano material
Safety evaluatio provides reference, and the most also the safe handling for nano zine oxide provides theory support.
Accompanying drawing explanation
Fig. 1 is three kinds of nano zine oxides (24 nm, 56 nm, 87 nm) transmission electron microscope (TEM) lab diagram;
Fig. 2 is that three kinds of nano zine oxides (24 nm, 56 nm, 87 nm) Malvern particle instrument detects grain size distribution;
Fig. 3 is normal 2D cellular morphology figure；
Fig. 4 is normal 3D cellular morphology figure；
Fig. 5 is the aspect graph after the 24nm nano zine oxide contact cultivation 12h of 2D Caco-2 cell and 25 g/ml；
Fig. 6 is the aspect graph after the 24nm nano zine oxide contact cultivation 12h of 3D Caco-2 cell and 25 g/ml；
Fig. 7 is the aspect graph after the 24nm nano zine oxide contact cultivation 12h of 2D Caco-2 cell and 100 g/ml；
Fig. 8 is the aspect graph after the 24nm nano zine oxide contact cultivation 12h of 3D Caco-2 cell and 100 g/ml；
Fig. 9 is vigour after three kinds of nano zine oxides (24,56,87 nm) contamination 2DCaco-2 cell 12 h；
Figure 10 is vigour after three kinds of nano zine oxides (24,56,87 nm) contamination 3DCaco-2 cell 12 h；
Figure 11 is IL-8 protein content after three kinds of nano zine oxides (24,56,87 nm) contamination 2D cell;
Figure 12 is IL-8 protein content after three kinds of nano zine oxides (24,56,87 nm) contamination 3D cell;
Figure 13 be 24 nm nano zine oxides contamination 2D with 3D cells after IL-8 protein content compare;
Figure 14 is the double dye figure of normal cell AO/EB；
Figure 15 be 10 g/ml 24 nm nano zine oxides contamination 2DCaco-2 cell 24 h after the double dye figure of AO/EB；
Figure 16 be 75 g/ml 24 nm nano zine oxides contamination 2DCaco-2 cell 24 h after the double dye figure of AO/EB；Figure 17 is 10
AO/EB double dye figure after 56 nm nano zine oxide contamination 2DCaco-2 cell 24 h of g/ml；
Figure 18 be 75 g/ml 56 nm nano zine oxides contamination 2DCaco-2 cell 24 h after the double dye figure of AO/EB；
Figure 19 be 10 g/ml 87 nm nano zine oxides contamination 2DCaco-2 cell 24 h after the double dye figure of AO/EB；Figure 20 is 75
AO/EB double dye figure after 87 nm nano zine oxide contamination 2DCaco-2 cell 24 h of g/ml；
Figure 21 is that 24 nm nano zine oxides are contaminated the percentage ratio of 2DCaco-2 cell difference death pathways after 24 h；
Figure 22 is that 24 nm nano zine oxides are contaminated the percentage ratio of 3DCaco-2 cell difference death pathways after 24 h.
Detailed description of the invention
Below in conjunction with the accompanying drawings, the present invention is described in further detail.
Experiment material:
Human colon adenocarcinoma's epithelium (Caco-2) cell Shanghai Chinese Hang Seng thing
Nano zine oxide Qinhuangdao Tai Ji ring
Hyclone company of the DMEM culture medium U.S.
MTT cell proliferation and the green skies of toxicity inspection test kit Beyotime()
Gibco company of phosphate buffer (PBS) U.S.
Hyclone (FBS) Hangzhou Ilex purpurea Hassk.[I.chinensis Sims
Hyclone company of 0.25% pancreatin (Typsin) U.S.
Human interleukin 8 (IL-8) detection kit peace enlightening is biological
Acridine orange (AO) U.S. Sigma subpackage
Ethidium bromide (EB) U.S. Sigma subpackage
Annexin V-Alexa Fluor 488 apoptosis detection kit Shanghai Mai Yueer
Other chemical reagent are analytical pure.
Key instrument:
Yin state, the ultrasonic washing instrument Ningbo City ultrasonic mechanical equipment factory of Frank
Power & light company of the High speed refrigerated centrifuge U.S.
Vacuum drying oven Shanghai Mei Puda Instrument Ltd.
Ultrapure water system Millipore company
Constant temperature digital display water-bath Shanghai Shen Yuan scientific instrument company limited
JEOL company of transmission electron microscope Japan
Thermo company of the carbon dioxide constant incubator U.S.
Tecan company of microplate reader Switzerland
Leica company of inverted fluorescence microscope Germany
Microson company of ultrasonic cell disruption instrument Germany
Vacuum drying oven superclean bench Suzhou purifies
Malvan company of Nano-S90 nano-particle size analysis Britain
Partech company of flow cytometer Germany
The Caco-2 cell based on 2D and the 3D evaluation methodology to nano zine oxide biological safety, comprises the steps:
1) 2D cell is cultivated: add Caco-2 cell strain containing 10% FBS and the complete DMEM culture medium of 1% L-glutaminate
In, 37 DEG C, 5% CO2 quiescent culture, a cell culture fluid was changed every 2 ~ 3 days, trophophase cell of taking the logarithm carries out follow-up examination
Test.
2) 3D cell is cultivated: is placed in by the Matrigel glue of sterile centrifugation tube subpackage before the test and dissolves 2 ~ 3 h on ice, then
Pipettor gun head and 96 well culture plates are placed in cooled on ice 1 h.Take 10 ~ 20 μ L Matrigel glue uniform embedding cell trainings
Support bottom plate, be placed in 37 DEG C of cell culture incubator 30 min and make it solidify；Digestion two dimension cell, counts and is diluted to 2 x 104/
ML, draws 50 μ L cell suspension and Matrigel glue respectively, proceeds to 96 well culture plates, then be placed in 37 DEG C thin after mixing by 1:1
Born of the same parents' incubator 30 min makes it solidify；Again the glue upper strata solidified drips 100 μ L and contains 10% FBS and 1% L-glutaminate
Complete DMEM culture medium, be 37 DEG C, 5% CO in condition2Incubator in quiescent culture, changed the most complete every 2 ~ 3 days
DMEM culture medium.
3) passage: Caco-2 cell attachment grows, and through 3-4 d, when cell grows to monolayer 80%-95%, discards supernatant
Liquid, rinses gently with appropriate PBS, discards waste liquid, adds 0.25% trypsin and carries out digesting 3-5 min, under inverted microscope
Observing when cell just becomes round, the DMEM culture medium adding a small amount of 10% hyclone terminates cell dissociation process, inhales with suction pipe
Taking culture medium, piping and druming makes cell the most adherent gently, is transferred in new culture bottle by cell suspension, and adds appropriate cultivation
Base, the condition of putting into is 37 DEG C, 5% CO2Incubator in cultivate after 3-4 d, carry out Secondary Culture next time.
4) nano-zinc oxide powder is dispersed in the DMEM culture medium of serum-free, makes variable concentrations nano zine oxide and hang
Liquid.Put in 4 DEG C of refrigerators standby.Because nano material is easily reunited so that this dispersion is unstable, makes the most every time
With front, ultrasonic cell disruption instrument need to be used to make nano material depolymerization, ultrasound condition is: 3 min, surpasses 3 s and stops 2 s, merit
Rate 300 w.
5) carry out morphological observation and cytoactive detect:
5.1) Characterization of Nano-materials: nano-particle, by the ultrasonic suspendible of dehydrated alcohol, is added drop-wise on copper mesh be dried mirror under transmission electron microscope
Inspection.Use Malvern particle instrument that nano-particle carries out intensity and the analysis of particle diameter distribution.
5.2) morphological observation: by the Caco-2 cell under 2D and 3D condition of culture, carry out contamination process, cell
With concentration be respectively 10 and 75 μ g/mL 24 nm nano zine oxides contamination solution co-culture 12 h.After hatching 12 h, it is being inverted
Basis of microscopic observation cellular morphology, and shoot photo.Cellular control unit is that the DMEM culture medium with serum-free co-cultures.
5.3) MTT colorimetric determination cytoactive is used: 2D and 3D cell is carried out contamination process, i.e. adds 0,10,
Tri-kinds of grain diameter nano zinc oxide contamination liquid of 24,56,87 nm of 25,50,75,100 μ g/ml variable concentrations are with cell altogether
Cultivate 12 h.After contamination time terminates, PBS rinse 2 times, every hole adds the MTT200 μ L of 0.05 mg/mL, carries out 4 h and hatch
After, absorb MTT supernatant, add 150 μ L DMSO and on shaking table, carry out low speed shake 15 min, for making intracellular formation
Purple crystal thing fully dissolves.The OD value at 570 nm is measured by multi-functional microplate reader.Test sets zeroing group (DMEM, MTT and
DMSO), matched group (cell, DMEM, MTT and DMSO).3D cultivates needs and sets without cell, comprises only the blank of gel
Group；
Calculating cell viability, formula is as follows:
Cell viability=OD (Aexp)-OD (Aneg)/OD (Acon)-OD (Aneg)
Wherein, Aexp is test group；Aneg is zeroing group；Acon is test control group.
5.4) inflammatory factor human interleukin-8(IL-8) mensuration:
(5.4.1) reagent is prepared:
Use IL-8 detection kit that IL-8 protein level is detected.The standard of reagent is carried out according to test kit description
Standby, draw standard curve；
(5.4.2) sample is prepared:
2D and 3D cell is carried out contamination process, i.e. adds three kinds of grain diameter nano zinc oxide of 0 ~ 100 μ g/mL variable concentrations
Contamination liquid and co-culture of cells 12 h.After contamination time terminates, 1000 rpm, centrifugal 10 min are to remove some polymer and to receive
Rice grain, collects supernatant and proceeds to 96 new orifice plates, pending follow-up test；
(5.4.3) sample is analyzed:
Microplate reader measures the OD value in each hole at 450 nm wavelength, calculates sample concentration.
5.5) the double dye method of AO/EB
Select to be in the Caco-2 cell of exponential phase, use 0.25% trypsinization, add appropriate DMEM immediately complete
Full culture medium, the cell suspension utilizing cell counting count board furnishing density to be 2 x 105/mL, it is inoculated in 6 well culture plates and cultivates 12 h
After, then co-culture 24 h with tri-kinds of grain diameter nano zinc oxide contamination solution of 24,56 and 87 nm that concentration is 10,75 μ g/mL.
After hatching 24 h, PBS rinse 2 times, add 300 μ L containing 100 ug/mL acridine oranges (AB) and 100 ug/mL ethidium bromides
(EB) PBS solution, with PBS rinse 2 times after dyeing 3min, at fluorescence microscopy Microscopic observation coloration result, and preservation of taking pictures.Right
It is that the DMEM culture medium with serum-free co-cultures according to group cell.
5.6) Annexin V-Alexa Fluor 488 method:
Under 2D condition of culture, carry out cell contamination process, i.e. add 24 nm nano oxygen of 0 ~ 100 μ g/mL variable concentrations
Change zinc contamination liquid and co-culture of cells 24 h；After contamination time terminates, collect floating cells and abandon upper strata nano zine oxide contamination
Liquid, PBS 2 times, cell, pending follow-up test are collected in digestion；
Under 3D condition of culture, carry out cell contamination process, i.e. add 24 nm nano zine oxide contamination liquid of variable concentrations with thin
Born of the same parents co-culture 24 h.After contamination time terminates, abandon upper strata nano zine oxide contamination liquid, add the 3D cell recovered liquid of 100 L,
Being swept by glue-line gently, is placed in and melts 30 min on ice, shakes up once every 10 min, treats that cell precipitation can sink to smoothly
At the bottom of pipe, illustrate that Matrigel gum base originally melts completely；Now, 2000 rpm, centrifugal 5 min, abandon supernatant, pending follow-up examination
Test；
The above-mentioned every hole of 2D and 3D cell sample is added the 1X Annexin-binding buffer re-suspended cell of 100 μ L.To
Every hole adds the 100 μ g/mL PI working solutions of 5 μ L Annexin V-Alexa Fluor 488 and 1 μ L respectively, at room temperature
Flow cytometer is utilized to detect fluorescent value at 530 nm and 575 nm after hatching 15 min immediately.
5.7) data process
Result uses SPSS19.0 statistical software to be analyzed, and data carry out one factor analysis of variance, calculates p < 0.05 and p
< the significance of difference of 0.01.It is at statistics that all data are represented as the meansigma methods ± SE of three independent trialss, P ＜ 0.05
The difference of meaning has significance.* represent p < 0.05(is compared with matched group), * * represent p < 0.01(is compared with matched group).
Experimental result and discussion
1) form and the particle diameter of nano material is distributed
After having children outside the state plan suspendible nano zine oxide sample such as Fig. 1 PBS as dispersant, under transmission electron microscope, observe three kinds of grain diameter nanos
Zinc oxide sample form, it is seen that its favorable dispersibility, some particles is coherent condition, and single-particle is many in bar-shaped or spherical.
Nano zine oxide sample particle diameter distribution such as tri-kinds of particle diameters of Fig. 2 is followed successively by 15-30 nm, 30-70 from left to right
nm、60-100 nm。
2) morphological observation result
Such as Fig. 3, matched group Caco-2 cell formed on 2D culture plate monolayer, adherent firmly, well-grown and in fusiformis or many
The cell model of dihedral.
Such as Fig. 4, cell aggregation growth in 3D culture model, formed bigger and in spherical cell mass.
After 24 nm nano zine oxide contamination 2D and the 3D cells of 25 g/mL, compare matched group, the shape of 2D cell in Fig. 5
State does not changes significantly, and adherent well but index of refraction is deteriorated, and gap becomes big, and part cell diminishes and rounded, 3D in Fig. 6
The form of cell is not changed in.When concentration of contamination increases to 75 g/mL, such as Fig. 7, visible in black in 2D culture model
And the nano zinc oxide particles reunited, 2D cell is in downright bad form and generation broken, adherent ability in various degree reduces, profile
Unintelligible, surface becomes " dirty ", quantity falls sharply, and in Fig. 8, the form of 3D cell is still without significantly change occurs, but cell pellets becomes
Little.
3) mtt assay detection cytoactive
Fig. 9 and Figure 10 is the cytoactive ratio after the nano zine oxide contamination Caco-2 2D and 3D cell 12 h of three kinds of particle diameters.As
Shown in Fig. 9, the successively decreasing in dose dependent with the rising of concentration of contamination of 2D cytoactive, the nano zine oxide of three kinds of particle diameters exists
The cytoactive of least concentration (10 g/mL) drops to 92.4%(24 nm respectively), 93.9%(56 nm), 92.1%(87 nm),
Compare matched group to be not significantly different from.Along with the increase (>=50 g/mL) of concentration of contamination, test group cytoactive seriously reduces,
There is compared with matched group the significance difference opposite sex (p < 0.01).Compare in organizing, when concentration is 50 g/mL, 24 nm, 56 nm
With the cell viability ratio respectively 64.5%, 68.7% and 72.1% of 87 nm nano zine oxides, 24 nm nano zine oxides compare 87nm
Zinc oxide cytoactive is relatively low and has significant difference (p < 0.05)；When concentration is 75 g/mL, the nano oxygen of three kinds of particle diameters
Changing zinc cell viability ratio respectively 38.8%, 49.8% and 55.3%, wherein the cell viability of 24 nm nano zine oxides is minimum, and 56
Nm and 87 nm nano zine oxides all have significant difference (p < 0.05) with it；When concentration is 100 g/mL, three kinds of nano oxygen
Changing the cell viability ratio respectively 21.4%, 32.3% and 41.2% of zinc, wherein the cell viability of 24 nm nano zine oxides is minimum, and three
Plant, between the nano zine oxide three of particle diameter, there is significant difference (p < 0.05) mutually.In a word, the nano zine oxide of three kinds of particle diameters
2D cytotoxicity size is respectively as follows: 24 nm > 56 nm > 87 nm.
As shown in Figure 10,3D cytoactive reduces with the rising of concentration of contamination, but compared with matched group, low concentration (≤
25 g/mL) under cell viability there is no significant difference therewith, concentration higher than the test group cell viability of 50 g/mL with right
Compare according to group and there is significant difference.Compare the size impact on cytoactive in organizing, find 24 nm, 56 nm and
The cell viability difference of 87 nm nano zine oxide test group does not has significance, shows that the size of nano zine oxide is thin to 3D
Cellular toxicity impact does not has notable difference.
The impact on cytoactive under 2D and 3D condition of culture of the nano zine oxide of relatively three kinds of particle diameters.With the highest contamination
As a example by concentration (100 g/mL), result shows: 24 nm, and the 2D cytoactive of 56 nm and 87 nm nano zine oxides is than phase comparison
76.9%, 65.8%, 56.6% is decreased respectively according to group；And 3D cytoactive ratio is compared matched group and is decreased 17.8% respectively,
16.3%, 13.3%.These results show 3D cytoactive apparently higher than 2D cell, so nano zine oxide is under 2D condition of culture
To Caco-2 cytotoxicity apparently higher than the cytotoxicity under 3D condition of culture.
4) mensuration of inflammatory factor IL-8
Figure 11 is that three kinds of grain diameter nano zinc oxide of variable concentrations compare with IL-8 protein content after 2D co-culture of cells 12 h.Three
Planting grain diameter nano oxidation zinc concentration less than 50 g/mL almost without causing IL-8 content to increase, concentration is the 56 of 75 g/mL
Nm nano zine oxide causes IL-8 content to dramatically increase.And concentration is when being 100 g/mL, IL-8 content increases at most, especially
24 nm nano zine oxides, IL-8 concentration is respectively 691 pg/mL(24 nm), 653 pg/mL(56 nm) and 617 pg/mL(87
Nm).When concentration is less than 75 g/mL, compare in organizing, find that the expression impact of IL-8 is not shown bright by grain size
Significant difference is different.
As shown in figure 12, after three kinds of grain diameter nano zinc oxide of variable concentrations and 3D co-culture of cells 12 h, IL-8 albumen
Content presents increasing trend along with the increase of concentration.After the especially 24 nano oxidized zinc concentrations of nm are higher than 75 g/mL,
IL-8 content dramatically increases；Compared with matched group, concentration is that 100 g/mL cause IL-8 content to add 2.5 times.Carry out in group
Relatively, after concentration is higher than 50 g/mL, 24 nm nano zine oxides cause the highest inflammatory reaction, and compare 56 nm nano oxygen
The contamination result changing zinc and 87 nm nano zine oxides shows that both do not have obvious difference.
As shown in figure 13, under 2D and 3D condition of culture, the concentration 24 nm nano zine oxide test group higher than 50 g/mL are drawn
The IL-8 protein expression risen has the significance difference opposite sex compared with matched group.When concentration of contamination is 50 and 100 g/mL, 3D cultivates
Under the conditions of IL-8 protein content compare and under 2D condition of culture, add 226 and 297 pg/mL respectively, show 24 nm nano oxygen
Change zinc is contaminated after 12 h, and 3D cell has more violent inflammatory reaction than 2D cell.Under 3D condition of culture, IL-8 albumen contains
Amount is to be incremented by the way of relying on concentration；Under 2D condition of culture, 24 nm nano zine oxides are only at maximum concentration (100 g/
ML) IL-8 protein content is caused to dramatically increase.
5) AO/EB double dye detection cell
Figure 14-20 is that under fluorescence microscope, Caco-2 cell is three kinds of grain diameter nano oxidations of 10 and 75 μ g/mL respectively with concentration
Zinc (24,56 and 87 nm) contamination solution co-cultures the metamorphosis after 24 h.VA represents that viable apoptotic cell, NVA represent late
Phase apoptotic cell, NVN represents non-viable non-apoptotic cell.P < 0.05, a represents that viable apoptotic cell is dramatically different with matched group, and b represents late
Phase apoptotic cell is dramatically different with matched group, and c represents that non-viable non-apoptotic cell is dramatically different with matched group.
Visible matched group is dyed to uneven green in a large number, there is the living cells (Figure 14) of normal configuration.Along with three kinds
Grain diameter nano oxidation zinc concentration raises, and living cells quantity significantly reduces, and the cell quantity of following three form is increasing, point
Not: cell membrane shrinkage, cytoplasm concentrates, and chromatin pyknosis spotting bulk or round bead shape, in homogeneous and bright green fluorescence
Viable apoptotic cell；Nucleus cracks and produces apoptotic body, or the late period of deflection dense poly-in orange-yellow or fluorescent red-orange
Apoptotic cell；Chromatin is sparse, and cell membrane is imperfect, and cell outline is unclear, in the non-viable non-apoptotic cell of uneven Chinese red fluorescence.This
A little results show that the increase of concentration of contamination makes cell that serious apoptosis to occur, and non-viable non-apoptotic cell quantity increases.It addition, along with nano oxidized
The reduction of zinc sample particle diameter, the cell quantity of the fluorescence that takes on a red color is increasing, is illustrating that the integrity of cell membrane sustains damage, make EB energy
Enough entering intracellular to dye nucleus, the quantity of non-viable apoptotic cell and non-viable non-apoptotic cell is being on the increase.Wherein, 24 nm
Nano zine oxide test group takes on a red color the cell quantity at most (Figure 17) of fluorescence, shows the number of non-viable apoptotic cell and non-viable non-apoptotic cell
Amount is at most.
6) Annexin V-Alexa Fluor 488 method
As shown in figure 21, concentration is 75 g/mL and 100 g/mL, and under 2D condition of culture, viable apoptotic cell is few, and ratio is divided
Not being 0.35%, under 0.28%, 3D condition of culture, viable apoptotic cell ratio is respectively 5.61%, and 7.42%.Concentration is 100 g/mL
Causing non-viable apoptotic cell and non-viable non-apoptotic cell under 2D condition of culture is 4.8 times and 33.1 times of matched group respectively；And 3D cultivates bar
Under part, non-viable apoptotic cell and non-viable non-apoptotic cell are 21.1 times and 2.5 times of matched group respectively.When concentration is 100 g/mL, 2D
Under condition of culture, normal live cells ratio is 14.7%, serious minimizing compared with matched group；Normal live cells number under 3D condition of culture
Relatively light shading is lacked, and ratio is 72.4%.As shown in figure 22, under 2D condition of culture, concentration causes cell to occur higher than 75 g/mL
Downright bad significantly more than apoptosis (p < 0.005)；Concentration mainly causes apoptosis less than 50 g/mL test group.But it is different
The nano zine oxide contamination 3D cell of concentration, the principal mode of cell death is all apoptosis.
Three kinds of grain diameter nano zinc oxide of the present invention refer to use 24, the nano zine oxide of 56,87 nm.
The present invention, with human colon carcinoma epithelium Caco-2 cell as object of study, first passes through transmission electron microscope and nano particle size instrument
Shape, size and distribution to nano zinc oxide particles characterize.Under 2D and 3D condition of culture, utilize inversion micro-
The contamination Caco-2 cell impact on its form of sem observation nano zine oxide, uses MTT colorimetric determination cytoactive, to carefully
Born of the same parents' inflammatory factor interleukin 8 (IL-8) detect, dead by the 2D cell after the double dye of fluorescence microscope AO/EB
Die form, simultaneously by the cell death way that flow cytomery is different.Nano zine oxide is to 2D Caco-2 cytotoxicity
Apparently higher than 3D cytotoxicity.Under 2D and 3D condition of culture, nano zine oxide is induction of the expression of inflammatory factor differentiation.3D
Cell is compared 2D cell and is responded significant lower to the toxicity of nano zine oxide.The research of the present invention demonstrates different cell and cultivates mould
Type interferes significantly on nano zine oxide to the biological effect of cell and the final assessment to nano oxidized zinc toxicity.
The present invention is illustrated according to embodiment, on the premise of without departing from present principles, if this device can also be made
Dry deformation and improvement.It should be pointed out that, the technical scheme that the modes such as all employing equivalents or equivalent transformation are obtained, all fall within this
In the protection domain of invention.
Claims (7)
1. the Caco-2 cell based on 2D and the 3D evaluation methodology to nano zine oxide biological safety, it is characterised in that include
Following steps:
1) 2D cell is cultivated: add Caco-2 cell strain containing 10% FBS and the complete DMEM culture medium of 1% L-glutaminate
In, 37 DEG C, 5% CO2 quiescent culture, a cell culture fluid was changed every 2 ~ 3 days, trophophase cell of taking the logarithm carries out follow-up examination
Test；
2) 3D cell is cultivated: is placed in by the Matrigel glue of sterile centrifugation tube subpackage before the test and dissolves 2 ~ 3 h on ice, then will move
Liquid device rifle head and 96 well culture plates are placed in cooled on ice 1 h；Take 10 ~ 20 μ L Matrigel glue uniform embedding cell culture plates
Bottom, is placed in 37 DEG C of cell culture incubator 30 min and makes it solidify；Digestion two dimension cell, counts and is diluted to 2 x 104/ mL, point
Do not draw 50 μ L cell suspension and Matrigel glue, proceed to 96 well culture plates after mixing by 1:1, then be placed in 37 DEG C of cells cultivations
Case 30 min makes it solidify；Again the glue upper strata solidified drips 100 μ L and contains the complete of 10% FBS and 1% L-glutaminate
DMEM culture medium, is 37 DEG C, 5% CO in condition2Incubator in quiescent culture, changed once completely DMEM every 2 ~ 3 days and cultivate
Base；
3) passage: Caco-2 cell attachment grows, and through 3-4 d, when cell grows to monolayer 80%-95%, discards supernatant
Liquid, rinses gently with appropriate PBS, discards waste liquid, adds 0.25% trypsin and carries out digesting 3-5 min, under inverted microscope
Observing when cell just becomes round, the DMEM culture medium adding a small amount of 10% hyclone terminates cell dissociation process, inhales with suction pipe
Taking culture medium, piping and druming makes cell the most adherent gently, is transferred in new culture bottle by cell suspension, and adds appropriate cultivation
Base, the condition of putting into is 37 DEG C, 5% CO2Incubator in cultivate after 3-4 d, carry out Secondary Culture next time；
4) nano-zinc oxide powder is dispersed in the DMEM culture medium of serum-free, makes variable concentrations nano zine oxide suspension；
Put in 4 DEG C of refrigerators standby；Before using, ultrasonic cell disruption instrument need to be used to make nano material depolymerization, ultrasonic bar every time
Part is: 3 min, surpasses 3 s and stops 2 s, power 300 w；
5) carry out Characterization of Nano-materials and morphological observation, use MTT colorimetric determination cytoactive, white to inflammatory factor
Cytokine-8 detects, by the 2D cell death form after the double dye of fluorescence microscope AO/EB, by fluidic cell
The cell death way that instrument detection is different；Use SPSS19.0 statistical software to be analyzed, data are carried out single factor test variance and divides
Analysis, calculates the significance of difference of p < 0.05 and p < 0.01.
2. the Caco-2 cell based on 2D and the 3D evaluation side to nano zine oxide biological safety as claimed in claim 1
Method, it is characterised in that
In step 5), described Characterization of Nano-materials, comprise the following steps: nano-particle, by the ultrasonic suspendible of dehydrated alcohol, is added drop-wise to
Microscopy under transmission electron microscope it is dried on copper mesh；Use Malvern particle instrument that nano-particle carries out intensity and the analysis of particle diameter distribution.
3. the Caco-2 cell based on 2D and the 3D evaluation side to nano zine oxide biological safety as claimed in claim 1
Method, it is characterised in that
In step 5), described morphological observation, comprise the following steps: by the Caco-2 cell under 2D and 3D condition of culture,
Carrying out contamination process, cell is that the 24 nm nano zine oxide contamination solution of 10 and 75 μ g/mL co-culture 12 h respectively with concentration；
After hatching 12 h, observation of cell form under inverted microscope, and shoot photo；Cellular control unit is the DMEM training with serum-free
Foster base co-cultures.
4. the Caco-2 cell based on 2D and the 3D evaluation side to nano zine oxide biological safety as claimed in claim 1
Method, it is characterised in that
In step 5), use MTT colorimetric determination cytoactive, comprise the following steps: 2D and 3D cell is carried out contamination process,
I.e. add tri-kinds of grain diameter nano zinc oxide dyes of 24,56,87 nm of 0,10,25,50,75,100 μ g/ml variable concentrations
Venom and co-culture of cells 12 h；After contamination time terminates, PBS rinse 2 times, every hole adds the MTT200 μ L of 0.05 mg/mL,
Carry out after 4 h hatch, absorbing MTT supernatant, add 150 μ L DMSO and on shaking table, carry out low speed shake 15 min, cell
The purple crystal thing of interior formation fully dissolves；The OD value at 570 nm is measured by multi-functional microplate reader；Test sets zeroing group:
DMEM, MTT and DMSO；Matched group: cell, DMEM, MTT and DMSO；3D cultivates needs and sets without cell, comprises only the sky of gel
White matched group；
Calculating cell viability, formula is as follows:
Cell viability=OD (Aexp)-OD (Aneg)/OD (Acon)-OD (Aneg)
Wherein, Aexp is test group；Aneg is zeroing group；Acon is test control group.
5. the Caco-2 cell based on 2D and the 3D evaluation side to nano zine oxide biological safety as claimed in claim 1
Method, it is characterised in that
In step 5), cross the 2D cell death form after the double dye of fluorescence microscope AO/EB, comprise the following steps:
Select to be in the Caco-2 cell of exponential phase, use 0.25% trypsinization, add appropriate DMEM immediately complete
Full culture medium, the cell suspension utilizing cell counting count board furnishing density to be 2 x 105/mL, it is inoculated in 6 well culture plates and cultivates 12 h
After, then co-culture 24 h with tri-kinds of grain diameter nano zinc oxide contamination solution of 24,56 and 87 nm that concentration is 10,75 μ g/mL；
After hatching 24 h, PBS rinse 2 times, add 300 μ L molten containing the PBS of 100 ug/mL acridine oranges and 100 ug/mL ethidium bromides
Liquid, with PBS rinse 2 times after dyeing 3min, at fluorescence microscopy Microscopic observation coloration result, and preservation of taking pictures；Cellular control unit is
Co-culture with the DMEM culture medium of serum-free.
6. the Caco-2 cell based on 2D and the 3D evaluation side to nano zine oxide biological safety as claimed in claim 1
Method, it is characterised in that
In step 5), inflammatory factor interleukin 8 is detected, comprises the following steps:
(1) reagent is prepared:
Use IL-8 detection kit that IL-8 protein level is detected；The standard of reagent is carried out according to test kit description
Standby, draw standard curve；
(2) sample is prepared:
2D and 3D cell is carried out contamination process, i.e. adds three kinds of grain diameter nano zinc oxide of 0 ~ 100 μ g/mL variable concentrations
Contamination liquid and co-culture of cells 12 h；After contamination time terminates, 1000 rpm, centrifugal 10 min are to remove some polymer and to receive
Rice grain, collects supernatant and proceeds to 96 new orifice plates, pending follow-up test；
(3) sample is analyzed:
Microplate reader measures the OD value in each hole at 450 nm wavelength, calculates sample concentration.
7. the Caco-2 cell based on 2D and the 3D evaluation side to nano zine oxide biological safety as claimed in claim 1
Method, it is characterised in that
In step 5), by the cell death way that flow cytomery is different, use Annexin V-Alexa Fluor
488 methods, comprise the following steps:
Under 2D condition of culture, carry out cell contamination process, i.e. add 24 nm nano oxygen of 0 ~ 100 μ g/mL variable concentrations
Change zinc contamination liquid and co-culture of cells 24 h；After contamination time terminates, collect floating cells and abandon upper strata nano zine oxide contamination
Liquid, PBS 2 times, cell, pending follow-up test are collected in digestion；
Under 3D condition of culture, carry out cell contamination process, i.e. add 24 nm nano zine oxide contamination liquid of variable concentrations with thin
Born of the same parents co-culture 24 h；After contamination time terminates, abandon upper strata nano zine oxide contamination liquid, add the 3D cell recovered liquid of 100 L,
Being swept by glue-line gently, is placed in and melts 30 min on ice, shakes up once every 10 min, treats that cell precipitation can sink to smoothly
At the bottom of pipe, illustrate that Matrigel gum base originally melts completely；Now, 2000 rpm, centrifugal 5 min, abandon supernatant, pending follow-up examination
Test；
The above-mentioned every hole of 2D and 3D cell sample is added the 1X Annexin-binding buffer re-suspended cell of 100 μ L；To
Every hole adds the 100 μ g/mL PI working solutions of 5 μ L Annexin V-Alexa Fluor 488 and 1 μ L respectively, at room temperature
Flow cytometer is utilized to detect fluorescent value at 530 nm and 575 nm after hatching 15 min immediately.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610477781.2A CN106086153A (en) | 2016-06-27 | 2016-06-27 | Caco 2 cell based on 2D and the 3D evaluation methodology to nano zine oxide biological safety |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610477781.2A CN106086153A (en) | 2016-06-27 | 2016-06-27 | Caco 2 cell based on 2D and the 3D evaluation methodology to nano zine oxide biological safety |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106086153A true CN106086153A (en) | 2016-11-09 |
Family
ID=57253054
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610477781.2A Pending CN106086153A (en) | 2016-06-27 | 2016-06-27 | Caco 2 cell based on 2D and the 3D evaluation methodology to nano zine oxide biological safety |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106086153A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108866147A (en) * | 2018-07-06 | 2018-11-23 | 宁波美晶医疗技术有限公司 | A kind of dynamic optical detection method of cells deformation and cell activity |
| CN112033878A (en) * | 2020-08-14 | 2020-12-04 | 桂林理工大学 | Method for determining influence of pollutants on active oxygen content in green alga cells |
| CN113846138A (en) * | 2021-09-13 | 2021-12-28 | 哈尔滨医科大学 | Biosafety evaluation method for delivering iron oxide nanoparticles through respiratory tract |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059961A (en) * | 2014-06-06 | 2014-09-24 | 中国计量学院 | Bio-safety evaluation method of nano zinc oxide based on Caco-2 cells |
| CN104195211A (en) * | 2014-06-06 | 2014-12-10 | 中国计量学院 | Method for evaluating biosecurity of 35 nm nano silver on the basis of intestinal epithelial cells |
| CN104195210A (en) * | 2014-06-06 | 2014-12-10 | 中国计量学院 | Method for evaluating biosecurity of 76 nm nano silver on base of intestinal epithelial cells |
-
2016
- 2016-06-27 CN CN201610477781.2A patent/CN106086153A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059961A (en) * | 2014-06-06 | 2014-09-24 | 中国计量学院 | Bio-safety evaluation method of nano zinc oxide based on Caco-2 cells |
| CN104195211A (en) * | 2014-06-06 | 2014-12-10 | 中国计量学院 | Method for evaluating biosecurity of 35 nm nano silver on the basis of intestinal epithelial cells |
| CN104195210A (en) * | 2014-06-06 | 2014-12-10 | 中国计量学院 | Method for evaluating biosecurity of 76 nm nano silver on base of intestinal epithelial cells |
Non-Patent Citations (3)
| Title |
|---|
| CHIA SING LING等: "《Biomimicry 3D Gastrointestinal Spheroid Platform for the Assessment of Toxicity and Inflammatory Effects of Zinc Oxide Nanoparticles》", 《SMALL》 * |
| DE BERARDIS BARBARA等: "《Exposure to ZnO nanoparticles induces oxidative stress and cytotoxicity in human colon carcinoma cells》", 《TOXICOLOGY AND APPLIED PHARMACOLOGY》 * |
| LEWINSKI NASTASSJA等: "《ytotoxicity of nano》", 《SMALL》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108866147A (en) * | 2018-07-06 | 2018-11-23 | 宁波美晶医疗技术有限公司 | A kind of dynamic optical detection method of cells deformation and cell activity |
| CN108866147B (en) * | 2018-07-06 | 2022-04-01 | 宁波美晶医疗技术有限公司 | Dynamic optical detection method for cell deformation and cell activity |
| CN112033878A (en) * | 2020-08-14 | 2020-12-04 | 桂林理工大学 | Method for determining influence of pollutants on active oxygen content in green alga cells |
| CN113846138A (en) * | 2021-09-13 | 2021-12-28 | 哈尔滨医科大学 | Biosafety evaluation method for delivering iron oxide nanoparticles through respiratory tract |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Souza et al. | Three-dimensional tissue culture based on magnetic cell levitation | |
| Moscato et al. | Poly (vinyl alcohol)/gelatin hydrogels cultured with HepG2 cells as a 3D model of hepatocellular carcinoma: a morphological study | |
| Simon et al. | In situ quantification of diverse titanium dioxide nanoparticles unveils selective endoplasmic reticulum stress-dependent toxicity | |
| CN106086153A (en) | Caco 2 cell based on 2D and the 3D evaluation methodology to nano zine oxide biological safety | |
| Wu et al. | Assessment of the toxicity and inflammatory effects of different-sized zinc oxide nanoparticles in 2D and 3D cell cultures | |
| Barosova et al. | An in vitro lung system to assess the proinflammatory hazard of carbon nanotube aerosols | |
| CN109735496A (en) | A kind of tumour cell chemotherapeutics three-dimensional resistant models and its method for building up | |
| Kathawala et al. | Emerging in vitro models for safety screening of high‐volume production nanomaterials under environmentally relevant exposure conditions | |
| CN104531620A (en) | Method for culturing lung cancer stem cells under 3D culture conditions | |
| Juarez-Moreno et al. | Monolayer (2D) or spheroids (3D) cell cultures for nanotoxicological studies? Comparison of cytotoxicity and cell internalization of nanoparticles | |
| Coccini et al. | Developmental neurotoxicity screening for nanoparticles using neuron-like cells of human umbilical cord mesenchymal stem cells: example with magnetite nanoparticles | |
| CN106119335A (en) | Caco 2 cell based on the 3D evaluation methodology to nano zine oxide biological safety | |
| De Simone et al. | Neuron-like cells generated from human umbilical cord lining-derived mesenchymal stem cells as a new in vitro model for neuronal toxicity screening: using magnetite nanoparticles as an example | |
| CN104195109B (en) | A kind of Lu-csf-1 and application thereof | |
| Marques et al. | Magnetic-based human tissue 3D cell culture: a systematic review | |
| Yan et al. | Effects of three different types of aloin on optical, mechanical, and antibacterial properties of waterborne coating on Tilia europaea surface | |
| CN107525920B (en) | Poly ion liquid magnetic nanocomposites and its application to trace enriching specificity of circulating tumor cell and detection | |
| Christ et al. | Sol-gel-derived fibers based on amorphous α-Hydroxy-Carboxylate-modified Titanium (IV) Oxide as a 3-dimensional scaffold | |
| ES2644243T3 (en) | 3D cell viability test | |
| Relucenti et al. | The ultrastructural analysis of human colorectal cancer stem cell-derived spheroids and their mouse xenograft shows that the same cells types have different ratios | |
| Phuangbubpha et al. | Optimizing THP-1 macrophage culture for an immune-responsive human intestinal model | |
| Khanal et al. | Nanotoxicity of nanodiamond in two and three dimensional liver models | |
| Revia et al. | High-Throughput Dispensing of Viscous Solutions for Biomedical Applications | |
| Zhao et al. | Comparative toxicity of nanomaterials to air-blood barrier permeability using an in vitro model | |
| CN104195210A (en) | Method for evaluating biosecurity of 76 nm nano silver on base of intestinal epithelial cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161109 |