CN111197068B - Method for drug sensitivity test based on super-hydrophobic microarray chip - Google Patents
Method for drug sensitivity test based on super-hydrophobic microarray chip Download PDFInfo
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Abstract
The invention provides a method for drug sensitivity test based on a super-hydrophobic microarray chip, which comprises the steps of culturing organoids in super-hydrophobic microarray chip micro-pits to obtain organoid cells, adding to-be-tested drugs with different concentrations and fluorescent dye solutions for cell proliferation and/or cell toxicity detection into the micro-pits, detecting the change of the fluorescent value of the fluorescent dye according to the cell proliferation and/or cell toxicity before and after the addition of the to-be-tested drugs with each concentration, and then determining the IC50 value of the to-be-tested drugs on the organoid cells according to the cell viability value of the to-be-tested drugs with each concentration. The invention combines the 3D culture technology with the super-hydrophobic microarray chip, can realize the culture of the organoids from different patients on the whole chip, and simultaneously performs the drug sensitivity test of various drugs on the organoids from one patient, and performs the drug sensitivity test of one or more drugs on the organoids from various patients.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method for carrying out drug sensitivity test based on a super-hydrophobic microarray chip.
Background
The super-hydrophobic microarray chip is based on PDMS or glass substrate, and comprises a micro-pit array and a micro cell culture chip with a super-hydrophobic surface layer between the micro-pits and adjustable micro-pit number. The arrangement mode, the size, the depth and the like of the micro-pit arrays in the chip can be adjusted, complete isolation among the micro-pit arrays can be realized, cross contamination among the micro-pits can be avoided, and good biocompatibility can be kept. Therefore, the chip can be used for 2D cell culture, 3D tumor organoid culture, in-vitro high-throughput drug screening, drug sensitivity detection and other bioanalysis.
Currently, drug screening and drug susceptibility testing for tumor organoids is mostly performed in well plates. Unlike cell lines under 2D culture conditions, tumor organoids are formed of 3D microspheres of different volume sizes that are grown by tumor cell aggregation. According to the literature report, the drug screening of a 384-well plate needs 15000-20000 tumor organoids in each ml of the initial amount to obtain an effective drug action curve. This is almost impossible to achieve with precious and small clinical cancer tissue samples. Therefore, for precious and small amount of samples, the samples need to be cultured, passaged and enriched before drug sensitivity test, which not only wastes time and labor and research cost, but also increases the drug resistance risk of tumor organoids through continuous passage,
disclosure of Invention
In order to solve the technical problems, the invention provides a method for carrying out drug sensitivity test based on a super-hydrophobic microarray chip, which is characterized by comprising the following steps: culturing organoid in a superhydrophobic microarray chip micro-pit to obtain organoid cells, adding to-be-tested drugs with different concentrations and a cell proliferation and/or cytotoxicity detection fluorescent dye solution into the micro-pit, detecting the change of the fluorescent dye according to the cell proliferation and/or cytotoxicity before and after the addition of each concentration of to-be-tested drug, and obtaining the cell viability value of each concentration of the to-be-tested drug according to the following formula,
wherein the ratio of CEV: cell viability values at each drug concentration;
TAB-1: cell viability value before adding medicine in each micro-pit of the organoid cell medicine adding group;
TAB-2: cell viability value after adding medicine in each micro-pit of the organoid cell medicine adding group;
VCAB-1: cell viability value before adding medicine into each micro-pit of the organoid cell blank control group, wherein organoid cells and culture medium are arranged in the micro-pits;
VCAB-2: organoid cell voidThe cell viability value after adding the medicine in each micro-pit of the control group is that organoid cells and culture medium are arranged in the micro-pit;
NCAB-1: detecting the cell viability value of the fluorescent dye empty matrigel before adding the drug in each micro-pit by cell proliferation and/or cell toxicity, wherein organoid cells and culture medium are not in the micro-pit;
NCAB-2: detecting the vitality value of the cells after being added with the drugs in each micro-pit of the fluorescent dye empty matrigel by cell proliferation and/or cell toxicity, wherein organoid cells and culture media are not arranged in the micro-pits;
n: number of micro-pits repeated per drug concentration;
and then determining the IC50 value of the test drug on the organoid cells according to the cell viability value of the test drug at each concentration.
In one embodiment, the method comprises the steps of:
step 1: inoculating the organoid suspension into a micro-pit on a super-hydrophobic microarray chip, spotting an organoid culture medium on a slide by using an automatic spotting instrument, aligning the slide with the chip by using a micro-aligner, adding the organoid culture medium into the micro-pit containing the organoid suspension, and culturing to obtain organoid cells;
step 2: scraping the culture medium on the matrigel in the micro-pit in the step 1, spotting the cell proliferation and/or cytotoxicity detection fluorescent dye working solution prepared by the cell proliferation and/or cytotoxicity detection fluorescent dye solution and the organoid culture medium on a slide by using an automatic spotting instrument, and aligning the slide carrying the working solution with the chip by using a micro-aligner; diluting the drugs to be tested into different concentrations, setting n times of repetition for each drug concentration, setting another group of negative controls, and using a group of fluorescent dye blank gel groups for cell proliferation and/or cytotoxic detection as background fluorescence controls; before adding drugs with different concentrations, scanning fluorescence in the chip micro-pits by using a fluorescence microscope to obtain TAB-1, VCAB-1 and NCAB-1;
and step 3: after the drug adding incubation is finished, scraping a culture medium containing drugs on the matrigel, spotting the cell proliferation and/or cell toxicity detection fluorescent dye working solution prepared by the cell proliferation and/or cell toxicity detection fluorescent dye solution and the organoid culture medium on a slide by using an automatic spotting instrument, aligning the slide carrying the working solution with the chip by using a micro-aligner, and scanning fluorescence in the chip micro-pits by using a fluorescence microscope to obtain TAB-2, VCAB-2 and NCAB-2; and
and 4, step 4: and obtaining cell viability values of the to-be-tested drugs under each concentration repetition, and fitting a curve to obtain the IC50 values of the to-be-tested drugs on the organoid cells.
In one embodiment, in step 1, the superhydrophobic microarray chip is immobilized in a petri dish, sterile water is added around the chip, and the petri dish is sealed with a sealing film.
In one embodiment, in step 1, the diameter of the micro-pits is 0.5-2mm and the depth is 100-300 μm.
In one embodiment, the micropits are 1mm in diameter and 200 μm in depth.
In one embodiment, each of said micro-pits is inoculated with 5-10 organoids.
In one embodiment, a sterile filter strip is used to scrape the medium off of the matrigel in the microwell.
In one embodiment, the cell proliferation and/or cytotoxicity detection fluorescent dye is alamar blue.
In one embodiment, the organoid is a tumor organoid.
In the invention, the 3D culture technology is combined with the super-hydrophobic microarray chip, so that organoids from different patients can be cultured on the whole chip, meanwhile, the organoids from one patient can be subjected to drug sensitivity test of multiple drugs, and the organoids from multiple patients can be subjected to drug sensitivity test of one or more drugs.
In the invention, the 3D micro-tumor cell viability detection reagent Almarblue (alamarBlue) is introduced, the sensitivity of the reagent is high, and about 5 tumor organoids in the micro-pores can be detected for viability. Based on this, we can do multiple of a few tumor organoids on the chipDrug sensitivity detection of species and obtaining a semi-lethal concentration (IC) by fitting a drug curve50) The result of the drug effect of various drugs for inhibiting the growth of tumor balls can be obtained one week after the surgical cancer tissues of the patient are obtained. The method of the invention has great practical significance for the development of new drugs and clinical medication.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic view of a process for fabricating a superhydrophobic microarray chip;
FIG. 2 is a schematic diagram of a high throughput superhydrophobic microarray chip in a petri dish;
FIG. 3 is a cross-sectional view of a high-throughput superhydrophobic microarray chip in a petri dish;
FIG. 4 is a flow chart of on-chip drug sensitivity testing;
FIG. 5 is a fluorescent scan of the chip before and after gefitinib addition; and
fig. 6 is a graph of cell viability for different concentrations of gefitinib.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, and not all examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example a fabrication of a superhydrophobic microarray chip (hereinafter referred to as a SMART chip) for drug sensitivity test according to the present invention.
And (3) slide silanization modification: aligning two clean slides and then horizontally placing the two clean slides on a desktop; adding about 200 mu L of silanization solution (10 mL: 80% (v/v) absolute ethyl alcohol and 20% (v/v)3- (trimethoxysilyl) propyl methyl methacrylate solution) along the gap between the two glass slides, fully mixing the two, and adjusting the pH value of the solution to 5.0 by using acetic acid until the gap between the two glass slides is completely filled with the solution; standing and modifying for 1h at room temperature; washing with anhydrous ethanol for 5min for 2-3 times; and (5) blowing the mixture by using nitrogen for later use.
The super-hydrophobic material comprises the following main components: composition of super hydrophobic polymer prepolymer solution (10 g): 24% wt BMA (butyl methacrylate, BMA)), 16% wt EDMA (ethylene glycol dimethacrylate, EDMA), 60% wt 1-decanol ((1-decanol)), 1% wt DMPAP (2,2-dimethoxy-2-phenylacetophenone, DMPAP)).
Preparation of the super-hydrophobic prepolymer: respectively weighing 600g of 1-decanol and 0.04g of catalyst DMPAP, adding into a 15mL centrifuge tube, wrapping with tinfoil paper, uniformly mixing for 1h at room temperature on a blood mixing instrument, then adding 2.4g of BMA and 1.6g of EDMA, and continuously mixing for 1h for later use.
Chip processing: as shown in fig. 1, the mold is placed on the bottom plate of the clamp, and the modified surface of the silanization modified glass slide is opposite to the mold and is clamped by a clamp; transferring the fixture, and injecting the super-hydrophobic prepolymer solution into the gap between the glass slide and the mold until the solution is completely filled; placing in an ultraviolet crosslinking box for ultraviolet exposure for 15-20 min; slightly prying the silanized glass slide and the mold by using a blade to obtain a micro-pit array of the super-hydrophobic layer with the thickness of about 200 mu m, namely a super-hydrophobic micro-array chip; washing the chip in absolute ethyl alcohol for 2-3 times; and (5) blowing the mixture by using nitrogen for later use.
And (3) sterilization: as shown in FIGS. 2 and 3, the bottom of the chip is coated with a layer Dow
3140 placing the gel in a 3.5cm culture dish, slightly pressing, standing for 15min to adhere the chip to the culture dish, immersing the culture dish with the chip in 75% ethanol for 15min, removing ethanol, and performing ultraviolet sterilization in a clean bench for 2 h. The specifications of the chip for drug sensitivity test are respectively 96 holes, 169 holes, 192 holes, 384 holes and 10 holes24 and 1188 wells to fit different initial amounts of tumor spheres.
EXAMPLE two drug sensitivity testing Using Alma blue on Superhydrophobic microarray chips
Transferring the prepared tumor organoid suspension (suspending the tumor organoids by using matrigel), organoid culture medium, sterile water, sterile chip (adhered to a culture dish as shown in figure 4-5-a) and sealing membrane into a sterile wet box at one time, and inoculating the matrigel mixed with organoids into each micro-pit on the chip according to a 300nL system per hole; after the matrigel had solidified slightly, organoid media was spotted onto the slides using an automatic spotting machine, 2 μ L per drop, as shown in fig. 4-5-b, and the slides were aligned to the chips using a microaligner to complete the plating. In order to avoid the evaporation of liquid drops in the chip micro-pits, 1mL of sterile water is required to be added around the chip in the culture dish, and the culture dish is sealed by a sealing film; culturing in a 5% CO2 incubator at 37 deg.C for 1-3 days, and replacing fresh culture medium every day.
First cell viability assay (AB-1): as shown in fig. 4-2, firstly preparing 1 x alamar blue working solution from alamar blue storage solution and culture medium according to a ratio of 1:9, spotting the working solution on a slide by using an automatic spotting instrument as shown in fig. 5-5-d, wherein each droplet is 300nL, then taking out the cultured tumor organoid chip, scraping the culture medium on matrigel in a pit on the chip by using a sterile filter paper strip, aligning the slide carrying the working solution with the chip by using a micro-aligner, sealing the culture dish by using a sealing film after completing the liquid feeding, placing the culture dish in a cassette, and incubating for 2-3h in a 37 ℃ and 5% CO2 incubator; after incubation, scanning fluorescence in the chip micro-pits by using a fluorescence microscope; after scanning, the gel drops were scraped off from the matrigel in the wells using a filter paper strip, and then 2. mu.L of fresh organoid medium was added to each well (see FIG. 4-1), and the wells were incubated at 37 ℃ in a 5% CO2 incubator.
As shown in fig. 4-3, the drugs were first diluted 5-fold in a sterile operating table by looking up the highest plasma concentration of the anti-tumor drug in humans, with 4 replicates per drug concentration, another set of 0.1% DMSO as a blank cell well control (VC), and another set of empty matrigel as a fluorescent alamar blue background control (NC).
The method comprises the steps of using an automatic sample applicator to sample a medicament (gefitinib) in a micropore of a chip, wherein the chip is a medicament-carrying chip (as shown in figure 4-5-c), placing the medicament-carrying chip in a culture dish, sealing the culture dish by a sealing film to ensure sterility, and placing the culture dish in a 4-DEG refrigerator for later use. Taking out the cultivation chip in step 2 from the incubator this moment, using aseptic filter paper strip to scrape off the liquid drop, using miniature aligner to aim at medicine carrying chip and cultivation chip, release medicine carrying chip and cultivation chip butt joint, accomplish and add the medicine process. The petri dish was sealed with a sealing film, placed in a dark box, and incubated at 37 ℃ in a 5% CO2 incubator. If the medicine needs to be replaced, the medicine carrying chip is taken away, and a new medicine carrying chip is covered according to the chip alignment flow.
Second cell viability assay (AB-2): after 3 days of drug action, a second cell viability assay (AB-2) was performed with reference to the first cell viability assay.
The results were processed and a curve was fitted to obtain drug IC50 values. As shown in FIG. 5, AB-1 and AB-2 are chip scans of cell viability assay performed before and after drug addition, fluorescence values in the micro-pits of the two cell viability assay scans were subjected to fluorescence-value conversion using ImageJ software, and calculated by the following formula,
TAB-1: cell viability value before adding medicine in each micro-pit of the organoid cell medicine adding group;
TAB-2: cell viability value after adding medicine in each micro-pit of the organoid cell medicine adding group;
VCAB-1: cell viability value before adding medicine into each micro-pit of the organoid cell blank control group, wherein organoid cells and culture medium are arranged in the micro-pits;
VCAB-2: the cell viability value after adding the medicine in each micro-pit of the organoid cell blank control group is that organoid cells and culture medium are arranged in the micro-pits;
NCAB-1: cell proliferation and/or cytotoxicity assaysMeasuring the cell viability value of the fluorescent dye empty matrigel before adding the drug into each micro-pit, wherein the micro-pit has no organoid cells and has a culture medium;
NCAB-2: detecting the vitality value of the cells after being added with the drugs in each micro-pit of the fluorescent dye empty matrigel by cell proliferation and/or cell toxicity, wherein organoid cells and culture media are not arranged in the micro-pits;
n: number of micro-pits repeated per drug concentration;
then, the values of IC50 of the drug were obtained by fitting the values of the concentration, table 1 shows the value of cell viability at each concentration, and fig. 6 is a graph of each point, and the value of IC50 of the drug was obtained by fitting.
Table 1: fluorescence-converted values for each of the craters in the AB-1 and AB-2 chip scans,
| gefitinib/(mu M) | AB-1 | AB-2 | NC-AB1 | NC-AB-2 |
| VC (concentration 0) | 22045.04 | 20505.27 | 5314.018 | 8185.327 |
| 20657.24 | 17534.48 | 5314.018 | 8185.327 | |
| 25402.69 | 20183.63 | 5314.018 | 8185.327 | |
| 0.002 | 20076.51 | 13952.08 | 5314.018 | 8185.327 |
| 21086.6 | 16813.27 | 5314.018 | 8185.327 | |
| 26179.21 | 35965.67 | 5314.018 | 8185.327 | |
| 0.01 | 17439.17 | 15752.31 | 5314.018 | 8185.327 |
| 16943.68 | 12892.19 | 5314.018 | 8185.327 | |
| 18773.64 | 28590.38 | 5314.018 | 8185.327 | |
| 0.05 | 18326.62 | 12016.62 | 5314.018 | 8185.327 |
| 20280.59 | 15019.69 | 5314.018 | 8185.327 | |
| 20353.81 | 17906.76 | 5314.018 | 8185.327 | |
| 0.25 | 15680.12 | 10051.39 | 5314.018 | 8185.327 |
| 17911.86 | 10687.49 | 5314.018 | 8185.327 | |
| 17627.9 | 12189.64 | 5314.018 | 8185.327 | |
| 1.25 | 19736.38 | 13364.31 | 5314.018 | 8185.327 |
| 18667.39 | 10788.68 | 5314.018 | 8185.327 | |
| 21202.03 | 11595.12 | 5314.018 | 8185.327 | |
| 6.25 | 21801.63 | 9929.157 | 5314.018 | 8185.327 |
| 18340.39 | 8769.447 | 5314.018 | 8185.327 | |
| 18550.72 | 9537.857 | 5314.018 | 8185.327 | |
| 31.25 | 21862.77 | 8938.956 | 5314.018 | 8185.327 |
| 21730.15 | 8639.64 | 5314.018 | 8185.327 | |
| 20467.7 | 8572.791 | 5314.018 | 8185.327 |
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims (9)
1. A method for performing a drug sensitivity test based on a superhydrophobic microarray chip for non-diagnostic purposes, the method comprising: culturing organoid in a superhydrophobic microarray chip micro-pit to obtain organoid cells, adding to-be-tested drugs with different concentrations and a cell proliferation and/or cytotoxicity detection fluorescent dye solution into the micro-pit, detecting the change of the fluorescent dye according to the cell proliferation and/or cytotoxicity before and after the addition of each concentration of to-be-tested drug, and obtaining the cell viability value of each concentration of the to-be-tested drug according to the following formula,
wherein the ratio of CEV: cell viability values at each drug concentration;
TAB-1: cell viability value before adding medicine in each micro-pit of the organoid cell medicine adding group;
TAB-2: cell viability value after adding medicine in each micro-pit of the organoid cell medicine adding group;
VCAB-1: cell viability value before adding medicine into each micro-pit of the organoid cell blank control group, wherein organoid cells and culture medium are arranged in the micro-pits;
VCAB-2: the cell viability value after adding the medicine in each micro-pit of the organoid cell blank control group is that organoid cells and culture medium are arranged in the micro-pits;
NCAB-1: detecting the cell viability value of the fluorescent dye empty matrigel before adding the drug in each micro-pit by cell proliferation and/or cell toxicity, wherein organoid cells and culture medium are not in the micro-pit;
NCAB-2: detecting the vitality value of the cells after being added with the drugs in each micro-pit of the fluorescent dye empty matrigel by cell proliferation and/or cell toxicity, wherein organoid cells and culture media are not arranged in the micro-pits;
n: number of micro-pits repeated per drug concentration;
then determining the IC of the drug to be tested on the organoid cells according to the cell viability value of the drug to be tested at each concentration50The value is obtained.
2. Method according to claim 1, characterized in that it comprises the following steps:
step 1: inoculating the organoid suspension into a micro-pit on a super-hydrophobic microarray chip, spotting an organoid culture medium on a slide by using an automatic spotting instrument, aligning the slide with the chip by using a micro-aligner, adding the organoid culture medium into the micro-pit containing the organoid suspension, and culturing to obtain organoid cells;
step 2: scraping the culture medium on the matrigel in the micro-pit in the step 1, spotting the cell proliferation and/or cytotoxicity detection fluorescent dye working solution prepared by the cell proliferation and/or cytotoxicity detection fluorescent dye solution and the organoid culture medium on a slide by using an automatic spotting instrument, and aligning the slide carrying the working solution with the chip by using a micro-aligner; diluting the drugs to be tested into different concentrations, setting n times of repetition for each drug concentration, setting another group of negative controls, and using a group of fluorescent dye blank gel groups for cell proliferation and/or cytotoxic detection as background fluorescence controls; before adding drugs with different concentrations, scanning fluorescence in the chip micro-pits by using a fluorescence microscope to obtain TAB-1、VCAB-1And NCAB-1；
And step 3: after the drug adding incubation is finished, scraping a culture medium containing drugs on the matrigel, spotting the cell proliferation and/or cell toxicity detection fluorescent dye working solution prepared by compounding the cell proliferation and/or cell toxicity detection fluorescent dye solution and the organoid culture medium on a slide by using an automatic spotting instrument, aligning the slide carrying the working solution with the chip by using a micro-aligner, and scanning fluorescence in the micro-pits of the chip by using a fluorescence microscope to obtain TAB-2、VCAB-2And NCAB-2(ii) a And
and 4, step 4: obtaining cell viability values of the to-be-tested drugs under each concentration repetition, and obtaining IC of the to-be-tested drugs on the organoid cells by fitting a curve50The value is obtained.
3. The method of claim 2, wherein in step 1, the superhydrophobic microarray chip is mounted in a petri dish, sterile water is added around the chip, and the petri dish is sealed with a sealing film.
4. The method as claimed in claim 2, wherein in step 1, the micro-pits have a diameter of 0.5-2mm and a depth of 100-300 μm.
5. The method of claim 4, wherein the micropits are 1mm in diameter and 200 μm deep.
6. The method of claim 2, wherein in step 1, each of said micro-pits is inoculated with 5-10 organoids.
7. The method of claim 2, wherein in step 2, a sterile filter strip is used to scrape the culture medium from the matrigel in the micro-wells.
8. The method of any one of claims 1 to 7, wherein the cell proliferation and/or cytotoxicity detecting fluorescent dye is alamar blue.
9. The method of claim 8, wherein the organoid is a tumor organoid.
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