CN102242181B - Flue gas condensate cytotoxicity determination method based on cell electronic sensor - Google Patents
Flue gas condensate cytotoxicity determination method based on cell electronic sensor Download PDFInfo
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Abstract
A flue gas condensate cytotoxicity determination method based on a cell electronic sensor is characterized by comprising the following technology steps:1) preparation of experiment reagents; 2) preparation of a flue gas condensate; 3) cell inoculation culture; 4) flue gas condensate contamination; 5) results and analysis. The invention utilizes a cell sensor chip to monitor cell growth conditions and influences of different condensate concentrations on cell growth after contamination of cells by a cigarette mainstream flue gas condensate on a real time basis, so as to establish a new, real-time, mark-free and high-flux method for cigarette mainstream flue gas cytotoxicity determination. Compared with a neutral red absorption method, the method of the invention omits a neutral red dyeing step and a complicated cleaning step to simplify a test process; meanwhile, quality control on cells is carried out according to a real-time cell growth curve after inoculation to optimize contamination time and contamination concentration, and monitor influences of different condensate concentrations on the cell growth curve after contamination of cells on a real time basis, so as to obtain a more accurate and reliable experiment result.
Description
Technical field
The present invention relates to the Cytotoxic determination techniques of cigarette mainstream flue gas condensation product, relate to specifically a kind of based on smoke condensate cytotoxic assay novel method real-time, unmarked, the high-flux cell sensor chip.
Background technology
The in vitro toxicology test is significant for the toxicity test that Human Health Risk assessment, cigarette Evaluation of Harmfulness and cigarette add material.Wherein, the neutral red test as one of CORESTA recommend method is the feasible vitro cytotoxicity testing method of accepting extensively.But the end point determination method is adopted in neutral red test, and choosing that put detection time has great effect to detected result.Toluylene red dyestuff itself also can't be eliminated the impact of cell state.In addition, toluylene red dyeing, a plurality of cleaning step have increased the possibility of introducing personal errors.
In real time, unmarked, high-flux cell sensing technology is microelectronics cell sensor chip to be incorporated into the surface be suitable for cell and attach bottom with the cell cultures microwell plate of growth, the adherent and propagation situation of the impedance variations near real-time quantitative monitoring cell by microelectrode.The impedance magnitude that the electronic cell sensing chip detects is scaled cytokine by instrument, characterizing the microelectrode superficial cell has or not and quantity, need not quantity and state that dye marker can reflect cell, the Real-time and Dynamic Detection that real realization is replied cytological effect.Compare with the toluylene red absorption process, this system has adopted non-marked, and undamaged detection technique can farthest avoid toluylene red dyeing, cleaning step for the interference of cytotoxicity detected result, and true reflection detects target compound toxicity.The electronic cell sensor has been simplified the cytotoxicity detecting step, need not other steps except cell inoculation and compound contamination, has reduced the impact of manual operation on detected result.
At present existing bibliographical information is used for the cytotoxic assay of different compounds and novel material with real-time cell sensor, and result and traditional MTT analytical method, neutral red uptake assay detected result are compared, and obtains good dependency.Proved and utilized feasibility real-time, unmarked, that the high-flux cell electronic sensor replaces conventional cell toxotest method.Yet there are no bibliographical information with the cytotoxicity analysis of electronic cell sensor application in cigarette smoke.Rely on automatic real time monitoring, unmarked, a series of advantages such as high-throughput, exploitation is measured novel method based on the smoke cytotoxicity of electronic cell sensor and is expected to obtain better accuracy and repeatability.
Summary of the invention
Purpose of the present invention is intended to overcome the prior art defective, simplify the cytotoxicity detecting step, need not the toluylene red dyeing, a kind of Cytotoxic novel method of electronic cell sensor determination cigarette mainstream flue gas condensation product of utilizing is provided, adherent, the propagation situation of the impedance variations near real-time quantitative monitoring cell that the method can be by electronic cell sensor microelectrode and contaminated by smoke condensate after cell state change.Automatically obtain the IC of smoke condensate by the cell growth curve that obtains after the contamination of different concns smoke condensate
50Value (half amount of suppression), the method is real-time, unmarked, high-throughput, to more accurate, the favorable reproducibility of the Cytotoxic mensuration of cigarette mainstream flue gas condensation product.
The objective of the invention is to be achieved through the following technical solutions:
The Cytotoxic measuring method of cigarette mainstream flue gas condensation product of the present invention comprises following processing step:
1. the preparation of smoke condensate: balance and smoking cigarette under the iso standard condition, flue gas total particulate matter (TPM) is collected by the Electrostatic pipe.Under nitrogen blows condition, methanol solvate is vapored away after utilizing methyl alcohol with flue gas total particulate matter wash-out, add at last the DMSO solvent of respective volume according to the TPM quality, obtain the standard reserving solution that ultimate density is 100 mg TPM/mL.Storing below-70 ℃ before using.
2. based on the smoke condensate cytotoxic assay of electronic cell sensor:
A, solution background deduction: at first in each hole of Tissue Culture Plate (E-plate) of electronic cell sensing chip has been integrated in the bottom, add 50 μ L substratum, then E-plate is placed cell culture case temperature to bathe 30 min, avoid because temperature variation causes the variation of impedance signal.E-plate after temperature bathed is positioned on the chip reader in the cell culture incubator, and the computer software by data analysis system begins the Real Time Monitoring impedance variations.At first the impedance signal that causes except 50 μ L substratum of system's bales catch changes background, and do not have inoculating cell in the culture plate this moment, so be 0 based on the cytokine (Cell Index) of impedance signal.The impedance signal of electrode depends on the characteristic of Cathode/Solution Interface, and is relevant with the Effective Conductivity of electrode surface character and solution of living in, with the volume-independent of electrode solution of living in.In the background deduction step, adding 50 μ L substratum does not have difference (data are unlisted) with the impedance signal that adds 100 μ L, the detection of 200 μ L same medium.
B, optimization cell inoculum density: the cell culture medium that at first adds 100 μ L in each hole of cell microelectronics sensing chip carries out the background deduction step, and deduction is because the microelectrode impedance variations that liquid nutrient medium causes.Then take out cell chip, inoculate respectively the CHO-K1 cell suspension of 100 μ L different concns in each hole, so that final inoculum density is 1000,2500,5000,10000, the CHO-K1 cell in 15000,20000,40000/hole, it is parallel that each concentration is set three holes.To only contain simultaneously the hole of 200 μ L substratum as the blank hole.Cell chip behind the inoculating cell is placed on the chip reader of cell culture incubator, the computer software by data analysis system carries out Real Time Monitoring to the adherent and propagation of cell.Determine the optimum cell inoculum density by the cell growth curve under the different cell inoculum densities.Final definite every hole adds the culture medium solution that 100 μ L contain 5000 CHO-K1 cells
Determining of C, contamination time: by the observation to cell growth curve in the many experiments, Chinese hamster ovary celI entered Exponential growth stage in about about 24 hours in inoculation as can be known, this moment cytokine (cell index, the CI value) reaches 1.0-1.3, expression cell proliferation is about 60% to the fraction of coverage of microwell plate floorage, is suitable for the smoke condensate contamination.
The optimization of D, DMSO solvent strength: smoke condensate contamination solution is scattered in the cigarette smoke granule phase substance in the DMSO solvent and prepares.In cytotoxicity experiment, for the cytotoxicity of true reflection smoke condensate, answer reduce DMSO Solvent effect.Cytotoxicity by investigating different concns DMSO solvent is as can be known: when endpoint detection (contaminating rear 24 hours), when the DMSO solvent strength is lower than 0.5%, the growth curve of cell growth curve and solvent control group does not have significant difference (P＜0.05), do not show obvious cytotoxicity, and when the DMSO solvent strength is higher than 0.75%, the growth curve of solvent control group and normal cell growth curve have significant difference (P＜0.05), show obvious cytotoxicity, therefore, in the final smoke condensate contamination solution, the DMSO solvent strength should not be higher than 0.5%, the cytotoxicity of solvent when as far as possible reducing to contaminate truly reflects the cytotoxicity of smoke condensate.
E, smoke condensate are contaminated: the smoke condensate storing solution that with concentration is 100mg TPM/mL is a series of concentration gradients with warm 37 ℃ of substratum dilutions of bathing, every hole adds the substratum that 50 μ L contain smoke condensate, so that final concentration of contamination is 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 150 μ g/mL, 200 μ g/mL, 250 μ g/mL, 300 μ g/mL, every Kongzui final volume is 200 μ L, parallel 6 holes of each concentration are measured, and DMSO solvent control group and Normal group (adding 50 μ L substratum) are set simultaneously.After the contamination E-plate is placed on the data reader of cell culture incubator the change of state in the 24h after the cell contamination is proceeded Real Time Monitoring.Present method adopts the method that adds successively 50 μ L substratum, 100 μ L cell suspensions, 50 μ L contamination solution to realize Cytotoxic mensuration, with other bibliographical informations contamination during step with culture hole in original substratum reject, the contaminating mode of being replaced by contamination solution is different.May there be the different defective of substratum reject degree in each culture hole in the replaced medium of bibliographical information for the contaminating mode of contamination solution, and the ultimate density that affects contamination solution owing to culture volume residual in the culture hole behind the reject is different is different.Therefore, present method adopts accurately control adding volume, the accurately method of strength of solution of guaranteeing to contaminate.
F, IC
50Obtaining of value: by the cell growth curve under the effect of different concns smoke condensate, the data analysis system of electronic cell sensor is obtained the IC of contamination this smoke condensate of rear any time automatically
50Value.Present method is with the IC of rear this smoke condensate that obtained in 24 hours of contaminating
50Value is as estimating the Cytotoxic standard of this smoke condensate.
The medium component that adopts among the present invention is the 2mM L-glutaminate, the F12K of 1.5 grams per liter sodium bicarbonates, 90%; Foetal calf serum, 10%.Cell culture condition is 37 ℃ of temperature, and environment is 95% air and 5% CO
2All through sterilization, whole experimentation is all finished in the aseptic technique environment for agents useful for same, equipment.
Method of the present invention has overcome the deficiency of existing cell toxicity determination method, utilizes the electronic cell sensor of energy Real Time Monitoring cell state to control the cell quality, optimized the conditions such as cell inoculum density, smoke condensate contamination time, smoke condensate concentration of contamination.Compared with prior art the inventive method has following excellent results:
1. utilize the electronic cell sensor can control the tenuigenin amount: because intrinsic sticking property, cellular form and the cell fission speed difference of cell, different culturing cell strains show diverse adhesion and growth performance graph.By drawing inferior strain (CHO-K1) specific of Chinese hamster ovary cell K1 performance graph, can realize the monitoring to the cell quality.
2. optimize the cell inoculum density: the growth curve by cell under the different vaccination density conditions can be optimized the cell inoculum density intuitively.When avoiding the cell inoculum density too high, produce contact inhibition between the cell after the inoculation, or the cell inoculum density crosses when low, cell proliferation speed is too slow after the inoculation.
3. accurate contamination time: in the cytotoxic assay test, contamination time need to enter the exponential growth after date at cell.Can intuitive judgment go out the time period that cell is in Exponential growth stage by the postvaccinal growth performance graph of Real Time Monitoring cell, be beneficial to determining of contamination time.
4. optimize DMSO solvent strength in the contamination solution: by the cytotoxicity optimization of the investigating different DMSO concentration DMSO solvent strength in the solution of finally contaminating, the cytotoxicity of DMSO solvent in the final smoke condensate of the reduce contamination solution truly reflects the cytotoxicity of smoke condensate.
5. optimize the concentration of contamination of smoke condensate: suitable concentration of contamination is for half amount of suppression (IC
50Value) accurate calculating is extremely important.Can intuitively understand the survival condition of cell under this concentration of contamination by the performance graph after the contamination of Real Time Monitoring cell, be conducive to determine fast suitable smoke condensate concentration of contamination.
6. present method has the simple advantage of operation steps, need not other steps except cell inoculation and smoke condensate contamination.
7. present method need not to add any indicator, can farthest avoid in traditional toxicity detection technique dyestuff toxicity for the interference of cytotoxicity detected result, can truly reflect to detect target compound toxicity.
8. present method has high-throughout advantage, can monitor simultaneously the cell state of 6 * 96 orifice plates.The present invention has the advantage that operates accurate, highly sensitive and good reproducibility.
Description of drawings
Fig. 1. measuring method schema of the present invention.
Fig. 2. the structural representation of electronic cell sensor.
Among Fig. 2: 1 is the E-plate Tissue Culture Plate, and 2 is the CO2 incubator, 3, the signal analysis reading system.
Fig. 3. the cell growth curve under the different cell inoculum densities.
Fig. 4. electronic cell sensor and mtt assay are to the quantitative analysis of cell.
Fig. 5. the optimization of DMSO solvent strength.
Fig. 6. the cellular change curve of electronic cell Sensor monitoring.
Fig. 7. the concentration logarithm that the electronic cell sensor is drawn-response value curve.
Embodiment
The present invention is described in further detail concrete technological process as follows below in conjunction with accompanying drawing:
1. the structure of electronic cell sensor: as shown in Figure 2 in the accompanying drawing, the electronic cell sensor is comprised of three parts: one is the electronic cell sensing chip, to insert the formula gold electrode to be incorporated into the culture plate bottom that is suitable for Growth of Cells and to make, when adding cell in culture plate, the impedance of culture plate bottom gold electrode is relevant with the propagation situation with adhering to of its superficial cell; Two is chip reader, and chip reader is positioned over CO
2In the cell culture incubator, be used for reading the impedance signal of each culture hole of electronic cell chip; Three is data analysis system, and chip reader links to each other by data tape with data analysis system, when the electronic cell sensing chip is placed on the chip reader, can read in real time and analyze impedance signal by data analysis system.
2. the optimization of cell inoculum density: Fig. 3 is the cell growth curve under the different vaccination density conditions of cell microelectronic sensor monitoring, and wherein CHO-K1 cell inoculum density is respectively 1000,2500,5000,10000,15000,20000, and 40000/hole.As shown in Figure 3, when the cell inoculum density was low, cell was very long latent period after the inoculation, and cell proliferation is slow, is unfavorable for Cytotoxic mensuration.When the cell inoculum density is too high, produce contact inhibition between the cell after the inoculation, can't observe obvious Exponential growth stage, and the impedance detection deviation of signal between the parallel hole is larger.When the cell inoculum density is 5000 cells/well, can clearly observe the different times of cell by curve: a. cell adhesion and extension stage; B. lag phase; C. exponential phase of growth; D. stationary phase and decline phase.Therefore selecting the optimum cell inoculum density in this experiment is 5000 cells/well.
3. the optimization of smoke condensate contamination time: in the cytotoxic assay test, contamination time need to enter the exponential growth after date at cell.Present method can intuitive judgment go out the time period that cell is in Exponential growth stage by the postvaccinal growth performance graph of Real Time Monitoring cell, is beneficial to determining of contamination time.By cell inoculum density among Fig. 3 be 5000 cells/well cell growth curve as can be known, after cell was inoculated about 26 hours, cell entered Exponential growth stage, inoculate about 80 hours after cell enter decline phase.Therefore determine that the smoke condensate contamination time is rear about 26 hours of inoculation, this moment cell has been in Exponential growth stage, and the observation period after the contamination all is in Exponential growth stage.
4. cell growth curve is used for the cell quality control: because intrinsic sticking property, cellular form and the cell fission speed difference of cell, different culturing cell strains show diverse adhesion and growth performance graph.Select the cell growth curve of 5000 cells/well among Fig. 3 to be the quality control curve of CHO-K1 cell in the experiment in this experiment.The quality control curve provides quality control standard for the cell between same experiment and different experiments, can operate and process correctly, exactly cell, rather than the supposition cell is in suitable the treatment stage.During each experiment, cell growth curve and quality control curve are compared, less than 10% standard as the cell quality control, the state of controlling each experimental cell is consistent as far as possible take the deviation of signal of same detection time point, improves the repeatability that cytotoxicity detects.In the experimentation of the present invention, the deviation of each experimental cell growth curve and quality control curve is all less than 10%.
5. the electronic cell sensor characterizes the Accuracy Verification of viable cell number: quantity and state that can the cytokine value that detect based on impedance signal accurately show viable cell are that the electronic cell sensor is used for the key that the smoke condensate cytotoxicity detects.Therefore by after the accurate cell counting, inoculate respectively the CHO-K1 cell in 172,497,2491,4000,6000,8000,10000/hole in electronic cell sensing chip and ordinary cells culture plate, it is 6 parallel that each inoculum density is set.The cell inoculation is after 6 hours, the cytokine and the mapping of cell inoculation number that obtain with the electronic cell sensor obtain Fig. 4 A, detect in the absorbance obtain and the ordinary cells culture plate cell inoculation number maps and obtains Fig. 4 B with mtt assay, detect as can be known the cytokine value that obtains by Fig. 4 A and become good linear relationship with the inoculating cell number, the electronic cell sensor can attach vegetative state and viable cell quantity by the accurate response cell.The electronic cell sensor is suitable with classical mtt assay to the accuracy of viable count flow measurement.
6. the optimization of solvent strength: smoke condensate contamination solution is scattered in the cigarette smoke granule phase substance in the DMSO solvent and prepares.In cytotoxicity experiment, for the cytotoxicity of true reflection smoke condensate, answer reduce DMSO Solvent effect.Fig. 5 has investigated the cytotoxicity of different concns DMSO solvent.As figure shows, with increasing of DMSO solvent strength, cytokine reduces degree and increases gradually.According to the statistical data analysis, when endpoint detection (contaminating rear 24 hours), when the DMSO solvent strength is lower than 0.5%, the growth curve of cell growth curve and solvent control group does not have significant difference (P＜0.05), do not show obvious cytotoxicity, and when the DMSO solvent strength is higher than 0.75%, the growth curve of solvent control group and normal cell growth curve have significant difference (P＜0.05), show obvious cytotoxicity, therefore, in the final smoke condensate contamination solution, the DMSO solvent strength should not be higher than 0.5%, the cytotoxicity of solvent when as far as possible reducing to contaminate truly reflects the cytotoxicity of smoke condensate.
7. the optimization of the concentration of contamination of smoke condensate: suitable concentration of contamination is most important for the Cytotoxic Accurate Determining of compound.Can intuitively understand the survival condition of cell under this concentration of contamination by the performance graph after the contamination of Real Time Monitoring cell, be conducive to determine fast suitable smoke condensate concentration of contamination.By great many of experiments as can be known, the concentration of contamination of smoke condensate is set as 25,50, and 100,150,200,250, during 300 μ g/mL, can satisfy the drafting of concentration logarithm-response value curve, guarantee half amount of suppression (IC
50Value) accurate calculating.
The present invention is described further below in conjunction with example, but is not restriction the present invention.
Example 1:
1. reagent and instrument:
Methyl-sulphoxide DMSO(biological reagent is pure, and purity is higher than 99%), the MTT dyestuff.The inferior strain of CHO-K1 Chinese hamster ovary cell is purchased from cell resource center of Shanghai Sheng Ke institute of the Chinese Academy of Sciences.The concrete composition of CHO-K1 cell culture medium is the 2mM L-glutaminate, the F12K of 1.5 grams per liter sodium bicarbonates, 90%; Foetal calf serum, 10%.XCELLigence electronic cell sensor (Roche Applied Science and ACEA Biosciences), 96 porocyte electronic sensor chips.AE163 electronic balance (sensibility reciprocal: 0.0001 g) (Switzerland Mettler company).
2. the preparation of smoke condensate storing solution
Balance cigarette sample A under the iso standard condition at first, according to FTC suction mode smoking cigarette, mainstream smoke total particulate matter (TPM) is collected by the Electrostatic pipe.Under nitrogen blows condition, methanol solvate is vapored away after utilizing methyl alcohol with flue gas total particulate matter wash-out, add at last the DMSO solvent of respective volume according to the TPM quality, obtain the standard reserving solution that ultimate density is 100 mg TPM/mL.Storing below-70 ℃ before using.
3. the preparation of different concns contamination solution:
The preparation of different concns smoke condensate is: dilute the smoke condensate storing solution of 100 mg TPM/mL with substratum, preparation flue gas condensing substrate concentration is 100,200,400,600, the culture medium solution of 800,1000,1200 μ g/mL.
4. Cytotoxic mensuration:
At first, every hole adds 50 μ L substratum on E-plate, after the system deduction solution background, by after the accurate cell counting on E-plate every hole add the substratum that 100 μ L contain 5000 Chinese hamster ovary celIs.Utilize the adherent and propagation situation of electronic cell sensor Real Time Monitoring cell.After about 24 hours, in the time of observing cell and enter Exponential growth stage, carry out the contamination of different concns smoke condensate.Utilize the smoke condensate of different concns that cell is contaminated, every hole adds the culture medium solution that 50 μ L contain smoke condensate, because 50 μ L are diluted to 200 μ L, so that the concentration of contamination of final smoke condensate is 25,50,100,150,200,250,300 μ g/mL, parallel 6 holes of each concentration are measured.Set simultaneously 6 parallel solvent control group and blank groups.Measurement result as shown in Figure 6, according to the different concns smoke condensate contamination cytokine value after 24 hours, system draws concentration logarithm-response value curve (Fig. 7) automatically, and provides the IC of cigarette sample A smoke condensate according to curve
50Value is 155.7 μ g/mL.
Example 2:
As described in Example 1, record the IC of cigarette sample B and sample C smoke condensate
50Value is respectively 94.7 μ g/mL and 115.9 μ g/mL.
Also use simultaneously CORESTA recommend method neutral red uptake assay and classical way mtt assay that the smoke condensate of cigarette sample A and B is carried out Cytotoxic evaluation, the detected result of three kinds of methods is as shown in table 1, such as table as can be known, the cytotoxic assay result of electronic cell sensor method is consistent with toluylene red method and mtt assay, but have fast, accurately, need not mark, step is easy and the advantage such as favorable reproducibility.
Claims (4)
1. smoke condensate cell toxicity determination method based on the electronic cell sensor is characterized in that: comprise following processing step:
(1), the preparation of smoke condensate: the preparation ultimate density is the standard reserving solution of 100 mg TPM/mL;
(2), based on the smoke condensate cytotoxic assay of electronic cell sensor:
A, solution background deduction: at first in each hole of Tissue Culture Plate of electronic cell sensing chip has been integrated in the bottom, add 50 μ L substratum, be positioned on the chip reader in the cell culture incubator, system's bales catch changes background except the impedance signal that is caused by substratum, thereby realizes cell is caused the monitoring of signal intensity;
B, cell inoculation: take out the Tissue Culture Plate that the electronic cell sensing chip has been integrated in the bottom, every hole adds the culture medium solution that 100 μ L contain 5000 CHO-K1 cells, place on the chip reader attaching and the vegetative state of the cytokine value Real-Time Monitoring cell that shows by data analysis system;
Determining of c, contamination time: when cell is in Exponential growth stage, carry out the smoke condensate contamination;
The optimization of d, DMSO solvent strength: the DMSO solvent strength is lower than 0.5%;
E, smoke condensate are contaminated: the smoke condensate storing solution that with concentration is 100mg TPM/mL is a series of concentration gradients with the substratum dilution, every hole adds the substratum that 50 μ L contain smoke condensate, so that final concentration of contamination is 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 150 μ g/mL, 200 μ g/mL, 250 μ g/mL, 300 μ g/mL, parallel 6 holes of each concentration are measured, and 0.3% DMSO solvent control group and the Normal group that adds 50 μ L substratum are set simultaneously; The Tissue Culture Plate of after the contamination aforesaid bottom having been integrated the electronic cell sensing chip places on the chip reader proceeds Real Time Monitoring to the change of state in the 24h after the cell contamination;
F, IC
50Obtaining of value: by the cell growth curve under the effect of different concns smoke condensate, the data analysis system of electronic cell sensor will be drawn concentration of contamination logarithm-response value curve automatically, obtain the IC of contamination this smoke condensate in the time of 24 hours
50Value.
2. the smoke condensate cell toxicity determination method based on the electronic cell sensor according to claim 1, it is characterized in that: the preparation method of smoke condensate is as follows: balance and smoking cigarette under the iso standard condition, flue gas total particulate matter (TPM) is collected by the Electrostatic pipe, under nitrogen blows condition, methanol solvate is vapored away after utilizing methyl alcohol with flue gas total particulate matter wash-out, add the DMSO solvent, the preparation ultimate density is the standard reserving solution of 100 mg TPM/mL.
3. the smoke condensate cell toxicity determination method based on the electronic cell sensor according to claim 1, it is characterized in that: medium component is the F12K:90% of 2mM L-glutaminate, 1.5 grams per liter sodium bicarbonates, foetal calf serum: 10%.
4. the smoke condensate cell toxicity determination method based on the electronic cell sensor according to claim 1, it is characterized in that: cell culture condition is 37 ℃ of temperature, environment is 95% air and 5% CO
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| CN105866200B (en) * | 2016-03-21 | 2018-04-10 | 杭州市红十字会医院 | A kind of device and method for Etoposide treatment effectiveness evaluation |
| WO2020104348A1 (en) * | 2018-11-20 | 2020-05-28 | F. Hoffmann-La Roche Ag | Method for seeding cells on a sensor surface |
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