WO2022241929A1 - Method for measuring maximum cross-sectional area of organoid, and application thereof - Google Patents
Method for measuring maximum cross-sectional area of organoid, and application thereof Download PDFInfo
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- WO2022241929A1 WO2022241929A1 PCT/CN2021/105299 CN2021105299W WO2022241929A1 WO 2022241929 A1 WO2022241929 A1 WO 2022241929A1 CN 2021105299 W CN2021105299 W CN 2021105299W WO 2022241929 A1 WO2022241929 A1 WO 2022241929A1
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Classifications
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
- G01B11/00—Measuring arrangements characterised by the use of optical techniques
- G01B11/28—Measuring arrangements characterised by the use of optical techniques for measuring areas
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Definitions
- the invention relates to the field of biomedicine, in particular, the invention relates to a method for detecting the maximum cross-sectional area of an organoid and its application.
- tumor treatment mainly involves surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy and endocrine therapy. About 70% of the patients need radiotherapy and chemotherapy, but due to individual differences in patients, tumor drug resistance or radiation resistance is an important factor restricting the successful treatment of tumors. At present, the overall effective rate of clinical tumor drugs is low (about 30%), and unnecessary and ineffective treatments for tumors cause huge waste. Therefore, the personalized treatment of tumor is imminent. Accelerating the research and development process of tumor clinical drugs and screening sensitive treatment groups for marketed tumor drugs are important fulcrums for changing the status quo of cancer treatment.
- the main tumor drug research and development models in the world include: tumor cell line model, patient tumor tissue allograft transplantation (PDX model).
- PDX model patient tumor tissue allograft transplantation
- the cost of 2D cell line screening is low and the efficiency is high, but the cell line is a single type of immortalization, which cannot reflect the heterogeneity, spatial structure and communication between various cells of the tumor.
- the human-derived tumor xenograft model can truly reflect the state of the tumor in the body and retain the heterogeneity of the tumor, there are species differences, long production cycle, high cost, etc., and many tumors have not yet been successfully established and cannot be established quickly. reflect the effect of medication. Since 2014, organoid technology has sprung up. Compared with mainstream drug research and development technologies, organoids have unparalleled advantages and advancements.
- Organoids are multicellular structures with a three-dimensional structure formed by using embryonic stem cells, pluripotent stem cells or adult somatic cells under a certain culture environment and the support of extracellular matrix, and are functionally close to organs.
- Tumor organoids Patient-derived tumor organoids, PDTOs
- Tumor organoids refer to a miniature three-dimensional 3D multicellular structure cultured in the laboratory from the primary tumor tissue of a tumor patient.
- tumor organoids Compared with 2D cell line culture, tumor organoids have a three-dimensional structure, which retains the heterogeneity of tumor tissue and is closer to the state of tumors in vivo.
- organoids Compared with xenograft tumor models, organoids have high efficiency and low cost, can better realize high-throughput drug screening, and more accurately and quickly simulate the real drug treatment response of patients.
- organoids are generally cultured on matrigel solids that are approximately hemispherical, there will be differences in location and ability to access growth factors, and organoids themselves have strong heterogeneity, resulting in their different shapes, sizes and distributions.
- the method for evaluating the size of organoids is mainly the image analysis method, which is to select a certain field of view under an optical or fluorescence microscope to take pictures of the organoids grown on Matrigel. Count the area and survival rate of each organoid in the output image, so as to judge the size of the organoid and cell survival after drug addition.
- Another approach is to grow organoids in specific hydrogel-coated microcavities so that they are as long as possible in one dimension and focal plane.
- the present invention is based on the inventor's discovery and recognition of the following facts and problems:
- the total maximum cross-sectional area of organoids was calculated after superimposing the tomographic images, and the drug efficacy results were measured according to the changes in the normalized value of the maximum total cross-sectional area of organoids in different drug treatment groups. match. Therefore, the method of calculating the area by continuous tomography of organoids can accurately evaluate the efficacy of tumor drugs.
- the present invention proposes a method for detecting the maximum cross-sectional area of an organoid, the method comprising: 1) performing visible light tomography processing on the organoid to be tested so as to obtain multiple tomographic images; Step 1) superimpose the obtained multiple tomographic images to obtain a superimposed image; and 3) obtain the maximum cross-sectional area of the organoid to be tested based on the superimposed images.
- the organoid image generated by the method for detecting the maximum cross-sectional area of the organoid is clear, and the number of organoids observed by taking only one cross-section is more accurate than the traditional method.
- the above use may further include at least one of the following additional technical features:
- the organoid to be tested is pre-cultured on Matrigel, and the Matrigel is hemispherical, and the visible light tomography is carried out under at least one of the following conditions: the volume of Matrigel and the imaging interval The ratio is 1 ⁇ L: 150 ⁇ m; the interlayer height is 48-52 ⁇ m or 98-102 ⁇ m; a dodging plate is added on the top of the porous plate; the LUT value is 50-55k.
- the smaller the layer height is set the more tomographic images are obtained, and the superimposed image can better reflect the real position and size of the organoid, but the smaller the layer height is, the longer it takes, and the computer and shooting machine The greater the loss; and problems such as uneven illumination, overexposure or too low exposure will lead to poor image effects, which is not conducive to the superposition of tomographic images.
- optical path, and exposure intensity were optimized; according to the optimized tomographic scanning parameters of the embodiment of the present invention, the obtained organoid image was clear, and the light intensity of the tomographic image was consistent, making the statistical results more accurate and the drug efficacy evaluation results more realistic.
- the visible light tomography is scanned with an inverted metallographic microscope under the condition of natural light projection or scanned with an inverted fluorescence microscope under the condition of staining the organoid with calcein.
- the organoid photographed by the inverted metallographic microscope can better distinguish the survival and death of the organoid, and obtain better statistical results.
- the organoids derived from patients have different shapes due to individual differences, and it is difficult to judge whether the organoids are dead or alive according to the morphology of the organoids taken under bright field conditions by an inverted metallographic microscope, you can choose to fluorescently label the organoids with calcein.
- the visible light tomography is not limited to the visible light microscope, and any visible light microscope that can accurately observe can be used.
- the excitation light is ultraviolet light.
- the wavelength of the ultraviolet light is 488nm.
- the present invention proposes a drug efficacy evaluation method, the method comprising: applying the drug to be screened to the organoid; The total maximum cross-sectional area of the organoid; based on the total maximum cross-sectional area of the organoid after administration, determine the normalized value of the total maximum cross-sectional area after administration of the organoid; and the normalized value based on the total maximum cross-sectional area of the organoid after administration , to determine whether the drug to be screened is the target drug; wherein, the maximum cross-sectional area of the organoid is obtained by the method described in the first aspect, and the total maximum cross-sectional area is the sum of the maximum cross-sectional areas of multiple organoids obtained and repeated The ratio of the number, the number of treatment repetitions is the number of preset repeated wells.
- the organoid image generated by using the method for detecting the maximum cross-sectional area of an organoid is clear, and the number of observable organoids is more accurate than taking a single cross-section, and the obtained evaluation results of the efficacy of tumor drugs are consistent with those obtained by patients after medication. The clinical responses were highly consistent.
- the above use may further include at least one of the following additional technical features:
- the number of the repeated holes is three.
- the organoids to be tested are pre-cultured on Matrigel, and the Matrigel is placed in the culture wells of a 48-well plate.
- the m culture wells are the same treatment.
- the number of repeated holes for this treatment is m, where m is an integer greater than 0.
- the normalized value of the total maximum cross-sectional area of the organoid after administration on the nth day is less than the normalized value of the total maximum cross-sectional area of the organoid on the 0th day after administration, which is the result of the to-be-treated
- the screening drug is an indication of the target drug, wherein, n is an integer not less than 1.
- the day when the organoid is administered is the 0th day;
- the standardized value is the total maximum cross-sectional area of the organoid on the nth day after administration and the total maximum cross-sectional area of the organoid on the 0th day after administration
- the normalized value of the total maximum cross-sectional area of the organoid after administration on day n is at least 30% less than the normalized value of the total maximum cross-sectional area of the organoid after administration on day 0, is
- the drug to be screened is an indication of the target drug.
- the drug to be tested is monitored for a 15-day standardized value of the maximum cross-sectional area of the organoid, wherein the normalized value of the organoid area is detected every 3 days. decrease, the tumor organoid area becomes smaller and the normalized value of the maximum cross-sectional area of the tumor organoid becomes smaller.
- the maximum cross-sectional area of the organoid is obtained and the normalized value of the total maximum cross-sectional area of the organoid is calculated.
- the change of the standardized value of the cross-sectional area is based on the fact that the obtained drug efficacy results have a high consistency with the clinical response of the patient after medication.
- the tomographic image is obtained from a plurality of different sections.
- the visible light progressive tomographic scanning is performed after setting the photographing top and bottom sections of the organoid hemispheric matrigel, wherein, the photographed section and the interlayer height can be set according to the experimental requirements, and the parameters of the tomographic image can be selected.
- the photographed section and the interlayer height can be set according to the experimental requirements, and the parameters of the tomographic image can be selected.
- the tomographic image is captured using Capture 2.1 software.
- the tomographic image is captured using the EDF depth-of-field expansion function in the Capture 2.1 software.
- the method is not limited to the shooting software, and any software that can accurately perform tomographic shooting can be used.
- the superimposed image is obtained by image processing based on light absorption values of multiple tomographic images and a wavelet algorithm.
- the method has no limitation on the software for obtaining superimposed images, and any software that can perform image processing based on the absorbance value of the tomographic image and the wavelet algorithm can be used.
- the superimposed image is superimposed and generated using Capture 2.1 software.
- the maximum cross-sectional area of the organoid is calculated using Image-Pro Plus 6.0 and/or Image J software.
- the method has no limitation on the software for calculating the maximum cross-sectional area of the organoid, and any software that can calculate the maximum cross-sectional area of the organoid in the image can be used.
- the change of the normalized value of the total maximum cross-sectional area of the organoid is calculated using GraphPad Prism software.
- the software used to make statistics on the changes in the normalized value of the total maximum cross-sectional area of organoids in the method there is no limitation on the software used to make statistics on the changes in the normalized value of the total maximum cross-sectional area of organoids in the method, and any software that can make statistics on the changes in the normalized value of the total maximum cross-sectional area of organoids can be used.
- the normalized value of the total maximum cross-sectional area of the organoid is the ratio of the total maximum cross-sectional area of the organoid on day n to the total maximum cross-sectional area of the organoid on day 0.
- the drug efficacy evaluation period of the organoid drug is 15 days.
- the tomographic images of the organoids are captured every 3 days starting from the 0th day in the drug efficacy evaluation period.
- the drug to be tested is monitored for 15 days of normalized value of the organoid area, wherein the normalized value of the organoid area is detected once every 3 days, and the obtained results of the drug efficacy of the intestinal cancer drug are consistent with the clinical response of the patient after medication. Very high consistency.
- the organoid is a tumor organoid.
- the tumor organoid is an intestinal cancer organoid.
- Fig. 1 is a graph showing the results of the determination of the efficacy of tumor drugs by the single-plane image analysis method of hydrogel organoids according to an embodiment of the present invention; the organoids are cultured in microcavity-coated petri dishes, and only one organoid exists in each well. organ. Add 0.001-20 ⁇ M Akt inhibitor Afuresertib for treatment, and take a single cross-section to count the cross-sectional area of each organoid. The abscissa on the right side is the type of drug added and the corresponding concentration, and the type of drug added is from left to right. For: DMSO (dimethyl sulfoxide), Afuresertib (Akt inhibitor), Gambogic acid (gambogic acid), the ordinate is the organoid area;
- Fig. 2 is a flow chart of intestinal cancer organoid culture according to an embodiment of the present invention
- the tumor tissue of a tumor patient undergoing surgery or biopsy is digested into a cell suspension, cultured in matrigel, and grown into a tumor organoid with a three-dimensional structure;
- Fig. 3 is an experimental design diagram of intestinal cancer organoids for drug experiments on intestinal cancer drugs according to an embodiment of the present invention.
- the organoids are planted in a 48-well plate, and when the diameter reaches 100 ⁇ m, 5-fluorouracil and captopor are respectively added to carry out the experiment.
- Treatment the culture medium was replaced every three days, and the culture period was 15 days;
- Fig. 4 is a flow chart of tomographic photography and an example diagram of superimposed images generated in the process of performing drug experiments on intestinal cancer drugs by intestinal cancer organoids according to an embodiment of the present invention
- Fig. 5 is a continuous tomographic scan of intestinal cancer organoids using an inverted metallographic microscope according to an embodiment of the present invention, and a trend diagram of the total maximum cross-sectional area changing with the interlayer height taken along the Z axis;
- Fig. 6 is a comparison diagram of the results of the statistical circles of the cross-sectional area of the intestinal cancer organoids according to the embodiment of the present invention using an inverted metallographic microscope for continuous tomographic scanning, and the z-axis shooting interlayer heights of 50 ⁇ m (left) and 150 ⁇ m (right); using ImageJ Carrying out circle selection, 150 ⁇ m loses many signals of small organoids compared with 50 ⁇ m;
- Fig. 7 is a comparison diagram of the tomographic images of intestinal cancer organoids observed before (left) and after (right) after changing the optical path by using an inverted metallographic microscope for continuous tomographic scanning of intestinal cancer organoids according to an embodiment of the present invention
- FIG. 8 is a comparison diagram of the tomographic images of intestinal cancer organoids observed under high exposure (left) and low exposure (right) conditions using an inverted metallographic microscope for continuous tomographic scanning of intestinal cancer organoids according to an embodiment of the present invention
- FIG. 9 is a trend diagram of the total maximum cross-sectional area of the colorectal cancer organoid according to an embodiment of the present invention using a fluorescence microscope for continuous tomographic scanning as the interlayer height changes along the Z-axis;
- Fig. 10 is a single focal plane photograph of intestinal cancer organoids on the 0th day (left) and 6th day (right) obtained by single-layer scanning with an inverted metallographic microscope according to an embodiment of the present invention
- Fig. 11 is the overlay of multiple Z-Stack focal planes on the 0th day (left) and the 6th day (right) obtained by continuous tomographic scanning of intestinal cancer organoids according to an embodiment of the present invention using an inverted metallographic microscope;
- Fig. 12 is a trend diagram of the total maximum cross-sectional area of organoids after treatment with 5-fluorouracil and Capto, obtained by continuous tomographic scanning of intestinal cancer organoids using an inverted metallographic microscope by planimetric method according to an embodiment of the present invention
- Fig. 13 is a tomographic overlay image of fluorescent labeling obtained by sequential tomographic scanning of intestinal cancer organoids using a fluorescence microscope using the planimetric method according to an embodiment of the present invention, where the dotted line circles represent a single organoid of various sizes and shapes;
- Fig. 14 is a trend diagram of the total maximum cross-sectional area of organoids after treatment with 5-fluorouracil and Capto, obtained by sequential tomographic scanning of intestinal cancer organoids using a fluorescence microscope using the planimetric method according to an embodiment of the present invention
- Fig. 15 is a fluorescent-labeled tomographic image obtained by using a high-power microscope for continuous tomographic scanning of intestinal cancer organoids using a three-dimensional modeling method according to an embodiment of the present invention; a total of 56 consecutive images were obtained in the shooting interval, and the three-dimensional organoids can be clearly observed Morphological changes, from no fluorescent signal at the beginning, to gradually photographing the upper half, center, and lower half of the organoid until the signal is lost;
- Fig. 16 is an example diagram obtained by using a high-power microscope to perform three-dimensional modeling on fluorescently labeled tomographic images obtained by continuous tomographic scanning of intestinal cancer organoids using a high-power microscope according to the three-dimensional modeling method of an embodiment of the present invention
- the upper left is a cross-sectional view in the X-axis direction
- the lower left is the vertical plane view where the largest section in the Z-axis direction is located
- the upper right is the longitudinal section view in the Y-axis direction
- the lower right is the result of organoid circle selection on the vertical plane where the largest cross-section in the Z-axis direction is located.
- Fig. 17 is a three-dimensional modeling method according to an embodiment of the present invention obtained by serial tomographic scanning of intestinal cancer organoids using a high-power microscope, and a trend diagram of the total surface area of the organoids after treatment with 5-fluorouracil and Capto; and
- Fig. 18 is a comparison chart of the evaluation results of the drug efficacy of the drugs 5-fluorouracil and Capto according to the colorectal cancer organoid area method according to the embodiment of the present invention and the response results of patients after clinical medication.
- Organoid drug efficacy results are divided into sensitive and tolerant (Note: Some human organs of patients have not been treated with Capto according to their clinical drug type, that is, patients have not been treated with Capto in clinical practice).
- the drug results of clinical patients are divided into two categories: good response and poor response. It can be seen from the figure that for patients with good clinical response, most of the organoid drug effects are sensitive, and for patients with poor response, most of the organoid drug effect results are tolerant.
- the drug efficacy measured by the planimetric method has a high consistency. The sensitivity was 78.01%, the specificity was 91.97%, and the accuracy was 84.43%.
- Embodiments of the present invention are described in detail below.
- the embodiments described below are exemplary only for explaining the present invention and should not be construed as limiting the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
- first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
- the features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
- “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
- intestinal cancer organoids are used as models, and advanced photomicrographing techniques are used to accurately determine the size change of organoids in Matrigel during the entire medication cycle after the addition of tumor drugs, thereby judging the efficacy of tumor drugs. guide clinical medication.
- the inventors scan with an inverted metallographic microscope or scan with an inverted fluorescence microscope under conditions where the organoids are stained with calcein. Only when the organoids are in good shape, the organoids photographed by the inverted metallographic microscope can better distinguish the survival and death of the organoids, and obtain better statistical results.
- organoids derived from patients have different shapes due to individual differences, and it is difficult to determine whether the organoids are alive or dead by the morphology of the organoids taken by an inverted metallographic microscope, you can choose to use calcein to fluorescently label the organoids. Calcein only labels living cells. Fluorescence microscopy was used to detect the maximum cross-sectional area of organoids. When the inverted fluorescent microscope is used for tomographic scanning, the excitation light is ultraviolet light; the wavelength of the ultraviolet light used is 488nm.
- the inverted metallurgical microscope can be used directly.
- Metallographic microscope for tomographic scanning; when the organoids derived from patients have different shapes due to individual differences, and it is difficult to judge whether the organoids are alive or dead by the morphology of the organoids taken by an inverted metallographic microscope, you can choose to fluorescently label the organoids with calcein. Chlorophyll only labels living cells, and the maximum cross-sectional area of organoids is detected using a fluorescence microscope.
- an overlay image is generated from the tomographic image in each culture well, and the position and maximum cross-sectional area of each organoid in Matrigel can be precisely found out by using the overlay image, and all the organs in a single culture well can be obtained.
- the collected fresh colon cancer biopsy tumor samples were photographed and the specific information was recorded, and after comparison, they were washed 5 times with pre-cooled PBS (adding 100 ⁇ penicillin/streptomycin), 5 min each time.
- PBS adding 100 ⁇ penicillin/streptomycin
- the digestive solution formula is: 7mL DMEM medium (GIBCO, C1199500BT), 500U/mL collagenase IV (Sigma-aldrich, C9407), 1.5mg/mL collagenase II (Solarbio, C8150), 20 ⁇ g/mL hyaluronidase ( Solarbio, h8030), 0.1 mg/mL dispase II (Sigma-aldrich, D4693), 10 ⁇ M RHOK inhibitor ly27632 (Sigma-aldrich, Y0503) and 1% fetal bovine serum. Digest for 40 minutes in a water bath shaker at 37°C.
- the culture plate was incubated in a constant temperature incubator at 37°C for 5-8 minutes and then taken out.
- the overall process is shown in Figure 2.
- Subculture once every 1-2 weeks.
- suck out the medium in the culture well add 500mL pre-cooled PBS, place the culture plate on the ice box for a while, blow off the matrigel with the pre-cooled pipette tip, transfer it into a 15mL sterile centrifuge tube and blow the matrigel completely Disperse and mix well, centrifuge at 69g at 4°C for 5min, and suck off the supernatant and Matrigel deposited at the bottom. Then add pre-cooled PBS and repeat washing three times to remove Matrigel.
- Organoids with good culture status were selected, digested and passaged into 48-well plates according to the method of Example 1.
- the initial inoculation density of organoids was 10-15/ ⁇ L Matrigel, and 300 ⁇ L medium was added to each well. Finally, 200 ⁇ 50 organoids were contained in 10 ⁇ L Matrigel in each well.
- the parameter optimization experiment was performed. A total of 3 replicate wells were set up.
- the shooting cycle was 15 days, starting from day 0 every 3 days Perform optical imaging of the organoids in each well, and use the EDF depth-of-field expansion function of the Capture 2.1 shooting software combined with an inverted metallographic microscope to perform progressive tomographic scanning and output multiple Z-Stack images.
- the shooting software will use the wavelet algorithm to superimpose multiple Z-Stack images based on the light absorption value of each layer to generate a complete superimposed image containing all the captured organoids.
- the specific operation process is shown in Figure 4.
- matrigel is a hemispherical solid different from cells, it is necessary to determine the top and bottom intervals of matrigel shooting, that is, where to start shooting and where to end. After optimizing the parameters, the inventors concluded that the imaging range of 10 ⁇ L of hemispherical matrigel is about 1500 ⁇ m, which is better.
- Inter-layer height that is, when tomography is taken, how many microns are selected in the shooting interval to take a photo to make the final output image the best.
- the inventors set the shooting layer heights of different Z axes as 10, 25, 50, 75, 100, and 150 ⁇ m, and the specific results are shown in Figures 5 and 6. It can be seen from Figure 5 that the total cross-sectional area of the obtained organoids is getting smaller and smaller as the interlayer height of the Z-axis photography increases, and the area statistical circle shown in Figure 6 shows that the interlayer height of 150 ⁇ m is compared with that of 50 ⁇ m.
- the condenser lens emits parallel light ⁇ , which forms light refraction inside the well plate and produces an included angle of ⁇ , resulting in uneven illumination. ; so that organoids near the edge of the well plate cannot be clearly observed. Therefore, the inventors changed the optical path by adding a uniform light plate on the top of the 48-well plate used to disrupt the parallel optical path and make it a uniform scattered light, as shown in FIG. 7 . In order to output accurate statistical images, it is also necessary to determine the optimal exposure to unify the light intensity used during superimposition. Overexposure or too low exposure will result in poor image quality and interfere with statistical results. After screening, the inventor found that setting the LUT (look up table) in the range of 50-55k works best, and the resulting image is shown in Figure 8.
- Organoids with good culture status were selected, digested and passaged into 48-well plates according to the method of Example 1.
- the initial inoculation density of organoids was 10-15/ ⁇ L Matrigel, and 300 ⁇ L medium was added to each well. Finally, 200 ⁇ 50 organoids were contained in 10 ⁇ L Matrigel in each well.
- the parameter optimization experiment was performed. A total of 3 replicate wells were set up.
- the shooting cycle was 15 days, starting from day 0 every 3 days Perform optical imaging of the organoids in each well, and use the EDF depth-of-field expansion function of the Capture 2.1 shooting software combined with an inverted fluorescence microscope to perform progressive tomographic scanning and output multiple Z-Stack images.
- the shooting software will use the wavelet algorithm to superimpose multiple Z-Stack images based on the light absorption value of each layer to generate a complete superimposed image containing all the captured organoids.
- the specific operation process is shown in Figure 4.
- the inventors used calcein to fluorescently label organoids, and calcein only labels living cells. Multiple organoids per well were labeled with calcein and fluorescently imaged every 3 days starting at day 0 during the 15-day culture period. Calcein with a final concentration of 4 ⁇ M was added to the medium for staining for 60 min, and the EDF depth-of-field expansion function of Capture 2.1 shooting software combined with an inverted fluorescence microscope was used to display green fluorescence pictures with 488 nm excitation light. The inventor screened the interlayer height of the Z-axis, and set the interlayer height to 25, 50, 100, 150, and 200 ⁇ m. The results showed that the imaging interval of 10 ⁇ L hemispherical matrigel was about 100 ⁇ m, and the remaining parameters were the same as those in the examples 2, the specific results are shown in Figure 9.
- the organoids obtained in Example 2 were used to obtain traditional single-view images and superimposed images obtained by tomographic scanning according to the parameters in Example 2 using an inverted metallographic microscope.
- the pictures obtained by single-layer shooting often contain many virtual focus organoids, which can not only observe the survival of organoids, but also bring difficulties to area statistics, which often leads to large errors in experimental results.
- a field of view where most of the organoids were in the same focal plane was found, and a single focal plane was used for shooting; on day 6, in the same field of view as day 0, the organoids were cultured and enlarged, and the focal plane Become inconsistent, as shown in Figure 10, organoids that cannot be focused will form ghost spots during imaging, resulting in the loss of the number of observable organoids, resulting in errors.
- Embodiment 5 area method evaluates drug efficacy accuracy verification
- Organoids with good culture status were selected, digested and passaged into 48-well plates according to the method of Example 1.
- the initial inoculation density of organoids was 10-15/ ⁇ L Matrigel, and 300 ⁇ L medium was added to each well. Finally, 200 ⁇ 50 organoids were contained in 10 ⁇ L of Matrigel per well, and drug treatment was performed when the diameter of organoids reached 100 ⁇ m.
- Two experimental groups including 5-fluorouracil (5-Fu) group and Capto (CPT-11) group.
- the organoids in each well treated with the two drugs 5-Fu and CPT-11 were optically imaged every 3 days from the 0th day, and the specific operation process and parameter settings were the same as in the example 2.
- the total maximum cross-sectional area of organoids on day 0 was normalized to 100%, and the ratio of the total maximum cross-sectional area of organoids counted on day 1 and subsequent days to the total maximum cross-sectional area on day 0 was the standardized value, that is, to obtain The percentage of the total maximum cross-sectional area of organoids at day 3 is shown in Table 2.
- the inventors used calcein to fluorescently label organoids, and calcein only labels living cells.
- Organoids from 3 wells per treatment were calcein-labeled and fluorescently imaged at 3-day intervals starting from day 0 for 15 days after dosing.
- Calcein with a final concentration of 4 ⁇ M was added to the culture medium of 3 wells for each detection and stained for 60 minutes, and the stained organoids were not used anymore;
- the EDF depth-of-field expansion function of the Capture 2.1 shooting software combined with an inverted fluorescence microscope was used to excite with 488nm Green fluorescence appeared, and the specific operation process and parameter settings were consistent with those in Example 3.
- the tomographic superimposed image of the obtained fluorescent marker is shown in FIG. 13 .
- the total maximum cross-sectional area of organoids on day 0 was normalized to 100%, and the ratio of the total maximum cross-sectional area of organoids counted on day 1 and subsequent days to the total maximum cross-sectional area on day 0 was the standardized value, that is, to obtain The percentage of the total maximum cross-sectional area of organoids at day 3, the specific results are shown in Table 3.
- the inventors used calcein to fluorescently label organoids, and calcein only labels living cells.
- Organoids from 3 wells per treatment were calcein-labeled and fluorescently imaged at 3-day intervals starting from day 0 for 15 days after dosing.
- Calcein with a final concentration of 4 ⁇ M was added to the culture medium of 3 wells for each detection and stained for 60 minutes, and the stained organoids were not used again; the organoids after fluorescent staining were combined with a high-power microscope that could filter stray light more than 40 times to analyze The organoids were tomographically photographed, as shown in Figure 15.
- Embodiment 6 area method measures drug effect and clinical drug effect consistency verification
- the inventors measured the sensitivity of 80 intestinal cancer patient-derived organoids to the clinical drugs 5-fluorouracil and captocin by using the planimetric method, and verified the consistency with the clinical drug responses of patients.
- the drug efficacy results of organoids are divided into sensitive and tolerant (Note: Some human organs of patients have not been treated with Capto according to their clinical drug type, that is, clinically patients have not used Capto).
- the drug results of patients are divided into two categories: good response and poor response; as can be seen from the figure, for patients with good clinical response, most of the organoid drug effects are sensitive, and for patients with poor response, most of the organoid drug effect results are tolerant.
- the drug efficacy measured by the planimetric method has a high consistency.
- the sensitivity was 78.01%, the specificity was 91.97%, and the accuracy was 84.43%.
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Abstract
A method for measuring the maximum cross-sectional area of an organoid, and an application thereof. The method comprises: 1) performing visible light tomography processing on an organoid to be measured, so as to obtain a plurality of tomographic images; 2) performing superimposition processing on the plurality of tomographic images obtained in step 1), so as to obtain a superimposed image; and 3) obtaining the maximum cross-sectional area of said organoid on the basis of the superimposed image.
Description
优先权信息priority information
本申请请求2021年05月17日向中国国家知识产权局提交的、专利申请号为202110535118.4的专利申请的优先权和权益，并且通过参照将其全文并入此处。This application claims priority and rights to the patent application No. 202110535118.4 filed with the State Intellectual Property Office of China on May 17, 2021, and is hereby incorporated by reference in its entirety.
本发明涉及生物医药领域，具体地，本发明涉及检测类器官最大截面积的方法及其应用。The invention relates to the field of biomedicine, in particular, the invention relates to a method for detecting the maximum cross-sectional area of an organoid and its application.
近年来，肿瘤逐渐成为我国人口的主要死亡原因。2017年约221万人因肿瘤去世，占当年所有死亡人数的24.8％。此外，肿瘤属于较典型的老年性疾病，随着我国人口老龄化程度的加剧，未来几十年内，预计因肿瘤死亡的人口比例会大幅增加，给社会带来巨大的压力和挑战。肿瘤治疗主要手术、放疗、化疗、靶向治疗、免疫治疗及内分泌治疗等手段。其中约70％的患者需要放化疗，但由于患者个体差异，肿瘤耐药或辐射抵抗是制约肿瘤成功治疗的重要因素。目前临床肿瘤药物整体有效率低(30％左右)，肿瘤不必要的治疗和没有效果的治疗造成巨大浪费。因此，肿瘤个性化治疗迫在眉睫。加速肿瘤临床药物的研发进程，为上市肿瘤药物筛选到敏感治疗人群，是改变肿瘤治疗现状的重要支点。In recent years, cancer has gradually become the main cause of death in our country. About 2.21 million people died of cancer in 2017, accounting for 24.8% of all deaths that year. In addition, cancer is a typical senile disease. With the aging of my country's population, the proportion of people who die from cancer is expected to increase significantly in the next few decades, bringing huge pressure and challenges to society. Tumor treatment mainly involves surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy and endocrine therapy. About 70% of the patients need radiotherapy and chemotherapy, but due to individual differences in patients, tumor drug resistance or radiation resistance is an important factor restricting the successful treatment of tumors. At present, the overall effective rate of clinical tumor drugs is low (about 30%), and unnecessary and ineffective treatments for tumors cause huge waste. Therefore, the personalized treatment of tumor is imminent. Accelerating the research and development process of tumor clinical drugs and screening sensitive treatment groups for marketed tumor drugs are important fulcrums for changing the status quo of cancer treatment.
目前国际上主要的肿瘤药物研发模型有：肿瘤细胞系模型、患者肿瘤组织异源移植即PDX模型。2D细胞系筛选成本低，效率高，但细胞系为永生化单一类型，不能反映肿瘤的异质性、空间结构以及各类细胞间的交流。人源肿瘤异体移植模型虽然可以比较真实的反映肿瘤在体内的状态，保留肿瘤的异质性，但其存在物种差异，制作周期长、成本高等问题，且很多肿瘤尚未成功建系且不能较快的反映用药效果。2014年以来，类器官技术异军突起。相比主流的药物研发技术，类器官具有无可比拟的优势和先进性。At present, the main tumor drug research and development models in the world include: tumor cell line model, patient tumor tissue allograft transplantation (PDX model). The cost of 2D cell line screening is low and the efficiency is high, but the cell line is a single type of immortalization, which cannot reflect the heterogeneity, spatial structure and communication between various cells of the tumor. Although the human-derived tumor xenograft model can truly reflect the state of the tumor in the body and retain the heterogeneity of the tumor, there are species differences, long production cycle, high cost, etc., and many tumors have not yet been successfully established and cannot be established quickly. reflect the effect of medication. Since 2014, organoid technology has sprung up. Compared with mainstream drug research and development technologies, organoids have unparalleled advantages and advancements.
类器官(Organoid)是利用胚胎干细胞、多能干细胞或成体体细胞，在一定的培养环境和细胞外基质的支撑作用下形成的具有三维结构的，在功能上接近器官的多细胞结构。肿瘤类器官(Patient-derived tumor organoids,PDTOs)是指取自肿瘤患者自身的原发性肿瘤组织在实验室中培养起来的一种微型立体3D多细胞结构。相比于2D细胞系培养，肿瘤类器官具有三维结构，保留了肿瘤组织的异质性，更接近体内肿瘤的状态。相比于异体移植瘤模型，类器官效率高，成本低，能更好地实现高通量药物筛选，更为精确迅速地模拟出患者真实的药物治疗响应情况。Organoids are multicellular structures with a three-dimensional structure formed by using embryonic stem cells, pluripotent stem cells or adult somatic cells under a certain culture environment and the support of extracellular matrix, and are functionally close to organs. Tumor organoids (Patient-derived tumor organoids, PDTOs) refer to a miniature three-dimensional 3D multicellular structure cultured in the laboratory from the primary tumor tissue of a tumor patient. Compared with 2D cell line culture, tumor organoids have a three-dimensional structure, which retains the heterogeneity of tumor tissue and is closer to the state of tumors in vivo. Compared with xenograft tumor models, organoids have high efficiency and low cost, can better realize high-throughput drug screening, and more accurately and quickly simulate the real drug treatment response of patients.
由于类器官一般在近似半球形的基质胶固体上培养，会存在位置以及接触生长因子能力的差异，且类器官本身具有很强的异质性，导致它们的形态大小和分布不一。目前评价类器官大小的方法主要是图像分析法，该方法是将生在基质胶上的类器官在光学或荧光显微镜下选取某个视野进行拍照。将输出图片中的每个类器官进行面积以及存活率统计，从而判断加药后类器官的大小和细胞存活情况。另外一种方法是让类器官生长在特定的水凝胶包被的微腔内，使他们尽可能长在一个维度和焦平面上。这一方法会大大降低类器官的异质性，从而拍摄单平面照片进行面积及存活统计，如图1所示，由于是3D培养模式，每个类器官所处的焦平面不同。因此形态不一以及可测量性有限，导致类器官难以进行高通量定量操作。目前已有的相对定量方法仅采用光学或荧光显微镜拍摄的一个截面进行统计，并不能精确的指示每个类器官的最大截面，因此不能很好的指示用药药效。为了更好的模拟体内用药效果，现有的方法都无法在保持类器官原有的状态下摆脱上述弊端，进行精确的药效分析。Because organoids are generally cultured on matrigel solids that are approximately hemispherical, there will be differences in location and ability to access growth factors, and organoids themselves have strong heterogeneity, resulting in their different shapes, sizes and distributions. At present, the method for evaluating the size of organoids is mainly the image analysis method, which is to select a certain field of view under an optical or fluorescence microscope to take pictures of the organoids grown on Matrigel. Count the area and survival rate of each organoid in the output image, so as to judge the size of the organoid and cell survival after drug addition. Another approach is to grow organoids in specific hydrogel-coated microcavities so that they are as long as possible in one dimension and focal plane. This method will greatly reduce the heterogeneity of organoids, so that single-plane photos can be taken for area and survival statistics. As shown in Figure 1, due to the 3D culture mode, the focal plane of each organoid is different. Therefore, the heterogeneous morphology and limited scalability make organoids difficult to perform high-throughput quantitative operations. The existing relative quantification methods only use a cross-section taken by an optical or fluorescence microscope for statistics, and cannot accurately indicate the maximum cross-section of each organoid, so they cannot indicate the drug efficacy well. In order to better simulate the drug effect in vivo, none of the existing methods can get rid of the above-mentioned drawbacks and perform accurate drug effect analysis while maintaining the original state of the organoid.
发明内容Contents of the invention
本发明是基于发明人对以下事实和问题的发现和认识作出的：The present invention is based on the inventor's discovery and recognition of the following facts and problems:
发明人发现，将类器官采用显微镜连续断层拍摄扫描后进行最大光强度投影，找出每个类器官的最大截面进行定量分析，可以很好的解决拍摄一个截面不能精确的指示每个类器官的最大截面积的难题；连续断层拍摄获得的叠加图像与传统单焦平面类器官拍摄图相比，图像更加清晰。将断层图像叠加后计算类器官总最大截面积，根据不同药物处理组的类器官总最大截面积标准化值的变化测出药物药效结果，所得结果与类器官测定药物药效的国标法结果相吻合。因此，类器官连续断层拍摄计算面积的方法可以准确的评估肿瘤药物的药效。The inventors found that the maximum light intensity projection is performed on the organoids using continuous tomographic photography and scanning of the microscope to find out the largest cross-section of each organoid for quantitative analysis, which can solve the problem that shooting a cross-section cannot accurately indicate each organoid. The problem of the largest cross-sectional area; the superimposed images obtained by continuous tomography are clearer than those obtained by traditional single focal plane organoids. The total maximum cross-sectional area of organoids was calculated after superimposing the tomographic images, and the drug efficacy results were measured according to the changes in the normalized value of the maximum total cross-sectional area of organoids in different drug treatment groups. match. Therefore, the method of calculating the area by continuous tomography of organoids can accurately evaluate the efficacy of tumor drugs.
在本发明的第一方面，本发明提出了一种检测类器官最大截面积的方法，所述方法包括：1)对待测类器官进行可见光断层扫描处理，以便获得多个断层图像；2)将步骤1)所获得的多个断层图像进行叠加处理，以便获得叠加图像；以及3)基于所述叠加图像，获得待测类器官的最大截面积。根据本发明的实施例，利用所述检测类器官最大截面积的方法生成的类器官图像清晰，相较于传统方法只拍摄一个截面观察到的类器官数量更加准确。In the first aspect of the present invention, the present invention proposes a method for detecting the maximum cross-sectional area of an organoid, the method comprising: 1) performing visible light tomography processing on the organoid to be tested so as to obtain multiple tomographic images; Step 1) superimpose the obtained multiple tomographic images to obtain a superimposed image; and 3) obtain the maximum cross-sectional area of the organoid to be tested based on the superimposed images. According to the embodiment of the present invention, the organoid image generated by the method for detecting the maximum cross-sectional area of the organoid is clear, and the number of organoids observed by taking only one cross-section is more accurate than the traditional method.
根据本发明的实施例，上述用途还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above use may further include at least one of the following additional technical features:
根据本发明的实施例，所述待测类器官预先培养于基质胶，所述基质胶呈半球状，所述可见光断层扫描是在以下条件的至少之一下进行的：基质胶的体积与拍摄区间的比值为1μL：150μm；层间高度为48～52μm或98～102μm；在多孔板顶部加入匀光板；LUT值为50～55k。根据本发明的实施例，层间高度设置越小获得的断层图像越多，叠加图像就越 能反应类器官的真实位置和大小，但层高设置越小，耗时越长，计算机及拍摄机器耗损越大；且光照不均匀、过度曝光或曝光太低等问题都会导致拍摄出的图像效果不佳，不利于进行断层图像的叠加，因此，发明人对可见光断层扫描的拍摄区间、层间高度、光路及曝光强度进行优化；根据本发明实施例优化后的断层扫描参数进行拍摄，获得的类器官图像清晰，断层图像的光强度一致，使得统计结果更加准确，药效评价结果更真实。According to an embodiment of the present invention, the organoid to be tested is pre-cultured on Matrigel, and the Matrigel is hemispherical, and the visible light tomography is carried out under at least one of the following conditions: the volume of Matrigel and the imaging interval The ratio is 1 μL: 150 μm; the interlayer height is 48-52 μm or 98-102 μm; a dodging plate is added on the top of the porous plate; the LUT value is 50-55k. According to the embodiment of the present invention, the smaller the layer height is set, the more tomographic images are obtained, and the superimposed image can better reflect the real position and size of the organoid, but the smaller the layer height is, the longer it takes, and the computer and shooting machine The greater the loss; and problems such as uneven illumination, overexposure or too low exposure will lead to poor image effects, which is not conducive to the superposition of tomographic images. , optical path, and exposure intensity were optimized; according to the optimized tomographic scanning parameters of the embodiment of the present invention, the obtained organoid image was clear, and the light intensity of the tomographic image was consistent, making the statistical results more accurate and the drug efficacy evaluation results more realistic.
根据本发明的实施例，所述可见光断层扫描是在自然光投射条件下，用倒置金相显微镜进行扫描或钙黄绿素对类器官染色的条件下用倒置荧光显微镜进行扫描。根据本发明的实施例，只有在类器官形态良好时，倒置金相显微镜拍摄的类器官才能较好的区分类器官的生存和死亡情况，得较好的统计结果。当病人来源的类器官因个体差异导致形态各异，根据倒置金相显微镜在明场条件下所拍摄类器官的形态很难判断死活时，可以选择用钙黄绿素对类器官进行荧光标记，钙黄绿素仅标记活细胞，利用荧光显微镜进行类器官最大截面积的检测。根据本发明实施例，当类器官形态良好时，所述可见光断层扫描对可见光显微镜并无限制，任何可准确进行观察的可见光显微镜均可使用。According to an embodiment of the present invention, the visible light tomography is scanned with an inverted metallographic microscope under the condition of natural light projection or scanned with an inverted fluorescence microscope under the condition of staining the organoid with calcein. According to the embodiment of the present invention, only when the shape of the organoid is good, the organoid photographed by the inverted metallographic microscope can better distinguish the survival and death of the organoid, and obtain better statistical results. When the organoids derived from patients have different shapes due to individual differences, and it is difficult to judge whether the organoids are dead or alive according to the morphology of the organoids taken under bright field conditions by an inverted metallographic microscope, you can choose to fluorescently label the organoids with calcein. Calcein Only live cells are labeled, and the maximum cross-sectional area of organoids is detected using a fluorescence microscope. According to the embodiments of the present invention, when the shape of the organoid is good, the visible light tomography is not limited to the visible light microscope, and any visible light microscope that can accurately observe can be used.
根据本发明的实施例，所述倒置荧光显微镜进行断层扫描时激发光为紫外光。According to an embodiment of the present invention, when the inverted fluorescence microscope performs tomography, the excitation light is ultraviolet light.
根据本发明的实施例，所述紫外光的波长为488nm。According to an embodiment of the present invention, the wavelength of the ultraviolet light is 488nm.
在本发明的第二方面，本发明提出了一种药物药效评价方法，所述方法包括：对类器官施加待筛选药物；基于类器官施药后的最大截面积，确认类器官施药后的总最大截面积；基于类器官施药后的总最大截面积，确定类器官施药后的总最大截面积的标准化值；以及基于所述类器官施药后的总最大截面积的标准化值，确定所述待筛选药物是否为目标药物；其中，类器官的最大截面积是通过第一方面所述的方法获得的，总最大截面积是获得的多个类器官最大截面积之和与重复数的比值，所述处理重复数为预先设置的重复孔的数量。根据本发明实施例，利用所述检测类器官最大截面积的方法生成的类器官图像清晰、可观察到的类器官数量较拍摄一个截面更加准确，得到的肿瘤药物药效评价结果与病人用药后的临床反应具有很高的一致性。In the second aspect of the present invention, the present invention proposes a drug efficacy evaluation method, the method comprising: applying the drug to be screened to the organoid; The total maximum cross-sectional area of the organoid; based on the total maximum cross-sectional area of the organoid after administration, determine the normalized value of the total maximum cross-sectional area after administration of the organoid; and the normalized value based on the total maximum cross-sectional area of the organoid after administration , to determine whether the drug to be screened is the target drug; wherein, the maximum cross-sectional area of the organoid is obtained by the method described in the first aspect, and the total maximum cross-sectional area is the sum of the maximum cross-sectional areas of multiple organoids obtained and repeated The ratio of the number, the number of treatment repetitions is the number of preset repeated wells. According to the embodiment of the present invention, the organoid image generated by using the method for detecting the maximum cross-sectional area of an organoid is clear, and the number of observable organoids is more accurate than taking a single cross-section, and the obtained evaluation results of the efficacy of tumor drugs are consistent with those obtained by patients after medication. The clinical responses were highly consistent.
根据本发明的实施例，上述用途还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above use may further include at least one of the following additional technical features:
根据本发明的实施例，所述重复孔的数量为3。根据本发明实施例，所述待测类器官预先培养于基质胶，基质胶置于48孔板的培养孔内，当m个培养孔进行相同处理时，这m个培养孔即为同一处理的重复孔，该处理的重复孔数量即为m，其中，m为大于0的整数。According to an embodiment of the present invention, the number of the repeated holes is three. According to an embodiment of the present invention, the organoids to be tested are pre-cultured on Matrigel, and the Matrigel is placed in the culture wells of a 48-well plate. When m culture wells are subjected to the same treatment, the m culture wells are the same treatment. Repeated holes, the number of repeated holes for this treatment is m, where m is an integer greater than 0.
根据本发明的实施例，第n天所述类器官施药后的总最大截面积的标准化值比第0天所述类器官施药后的总最大截面积的标准化值减少，是所述待筛选药物为目标药物的指示，其中，n为不小于1的整数。根据本发明实施例，类器官施药当天为第0天；标准化值为第n天所述类器官施药后的总最大截面积与第0天所述类器官施药后的总最大截面积的比值， 如，第1天类器官总最大截面积的标准化值为：S1＝第1天所述类器官施药后的总最大截面积/第0天所述类器官施药后的总最大截面积，S2＝第2天所述类器官施药后的总最大截面积/第0天所述类器官施药后的总最大截面积，S3＝第3天所述类器官施药后的总最大截面积/第0天所述类器官施药后的总最大截面积。According to an embodiment of the present invention, the normalized value of the total maximum cross-sectional area of the organoid after administration on the nth day is less than the normalized value of the total maximum cross-sectional area of the organoid on the 0th day after administration, which is the result of the to-be-treated The screening drug is an indication of the target drug, wherein, n is an integer not less than 1. According to the embodiment of the present invention, the day when the organoid is administered is the 0th day; the standardized value is the total maximum cross-sectional area of the organoid on the nth day after administration and the total maximum cross-sectional area of the organoid on the 0th day after administration For example, the normalized value of the total maximum cross-sectional area of the organoid on day 1 is: S1=the total maximum cross-sectional area of the organoid on day 1 after administration/the total maximum cross-sectional area of the organoid on day 0 after administration Cross-sectional area, S2=the total maximum cross-sectional area of the organoid on the 2nd day after administration/the total maximum cross-sectional area of the organoid on the 0th day after administration, S3=the organoid on the third day after administration Total maximum cross-sectional area/total maximum cross-sectional area of the organoid on day 0 after administration.
根据本发明的实施例，第n天所述类器官施药后的总最大截面积的标准化值比第0天所述类器官施药后的总最大截面积的标准化值减少至少30％，是所述待筛选药物为目标药物的指示。根据本发明实施例，对待测药物进行15天类器官最大截面积标准化数值监测，其中，类器官面积标准化数值每隔3天检测一次，如果由于药物的药效作用而导致癌细胞灭忙、活性降低，则会出现肿瘤类器官面积变小、肿瘤类器官最大截面积标准化数值变小的结果，经过验证，得到类器官最大截面积后计算类器官总最大截面积标准化值，以类器官总最大截面积标准化值的变化为依据所得到的药物药效结果与病人用药后的临床反应具有很高的一致性。According to an embodiment of the present invention, the normalized value of the total maximum cross-sectional area of the organoid after administration on day n is at least 30% less than the normalized value of the total maximum cross-sectional area of the organoid after administration on day 0, is The drug to be screened is an indication of the target drug. According to an embodiment of the present invention, the drug to be tested is monitored for a 15-day standardized value of the maximum cross-sectional area of the organoid, wherein the normalized value of the organoid area is detected every 3 days. decrease, the tumor organoid area becomes smaller and the normalized value of the maximum cross-sectional area of the tumor organoid becomes smaller. After verification, the maximum cross-sectional area of the organoid is obtained and the normalized value of the total maximum cross-sectional area of the organoid is calculated. The change of the standardized value of the cross-sectional area is based on the fact that the obtained drug efficacy results have a high consistency with the clinical response of the patient after medication.
根据本发明的实施例，所述断层图像是从互不相同的多个截面取得的。根据本发明实施例，设置类器官半球基质胶的拍摄顶部和底部区间后进行可见光递进式断层拍摄扫描，其中，所拍摄的区间以及层间高度可依据实验需求自行设置参数，选择断层图像的拍摄截面时应保证在图像清晰的情况下该截面可以拍摄到尽量多的类器官，以最大程度还原类器官的位置和大小。According to an embodiment of the present invention, the tomographic image is obtained from a plurality of different sections. According to the embodiment of the present invention, the visible light progressive tomographic scanning is performed after setting the photographing top and bottom sections of the organoid hemispheric matrigel, wherein, the photographed section and the interlayer height can be set according to the experimental requirements, and the parameters of the tomographic image can be selected. When shooting a cross-section, it should be ensured that as many organoids as possible can be photographed in the cross-section when the image is clear, so as to restore the position and size of the organoids to the greatest extent.
根据本发明的实施例，所述断层图像是使用Capture 2.1软件拍摄。According to an embodiment of the present invention, the tomographic image is captured using Capture 2.1 software.
根据本发明的实施例，所述断层图像是使用Capture 2.1软件中的EDF景深扩展功能拍摄。根据本发明实施例，所述方法对拍摄软件并无限制，任何可准确进行断层拍摄的软件均可使用。According to an embodiment of the present invention, the tomographic image is captured using the EDF depth-of-field expansion function in the Capture 2.1 software. According to the embodiment of the present invention, the method is not limited to the shooting software, and any software that can accurately perform tomographic shooting can be used.
根据本发明的实施例，所述叠加图像是基于多张断层图像的吸光值和小波算法进行图像处理获得的。根据本发明实施例，所述方法对获得叠加图像的软件并无限制，任何可基于断层图像的吸光值和小波算法进行图像处理的软件均可使用。According to an embodiment of the present invention, the superimposed image is obtained by image processing based on light absorption values of multiple tomographic images and a wavelet algorithm. According to the embodiment of the present invention, the method has no limitation on the software for obtaining superimposed images, and any software that can perform image processing based on the absorbance value of the tomographic image and the wavelet algorithm can be used.
根据本发明的实施例，所述叠加图像是使用Capture 2.1软件叠加生成。According to an embodiment of the present invention, the superimposed image is superimposed and generated using Capture 2.1 software.
根据本发明的实施例，所述类器官最大截面积是使用Image-Pro Plus 6.0和/或Image J软件计算。根据本发明实施例，所述方法对类器官最大截面积计算的软件并无限制，任何可对图像中类器官进行最大截面积计算的软件均可使用。According to an embodiment of the present invention, the maximum cross-sectional area of the organoid is calculated using Image-Pro Plus 6.0 and/or Image J software. According to the embodiment of the present invention, the method has no limitation on the software for calculating the maximum cross-sectional area of the organoid, and any software that can calculate the maximum cross-sectional area of the organoid in the image can be used.
根据本发明的实施例，所述类器官总最大截面积标准化值的变化是使用GraphPad Prism软件统计。根据本发明实施例，所述方法对类器官总最大截面积标准化值的变化进行统计的软件并无限制，任何可对类器官总最大截面积标准化值的变化进行统计的软件均可使用。According to an embodiment of the present invention, the change of the normalized value of the total maximum cross-sectional area of the organoid is calculated using GraphPad Prism software. According to the embodiment of the present invention, there is no limitation on the software used to make statistics on the changes in the normalized value of the total maximum cross-sectional area of organoids in the method, and any software that can make statistics on the changes in the normalized value of the total maximum cross-sectional area of organoids can be used.
根据本发明的实施例，所述类器官总最大截面积的标准化值为类器官第n天的总最大 截面积与第0天类器官总最大截面积的比值。According to an embodiment of the present invention, the normalized value of the total maximum cross-sectional area of the organoid is the ratio of the total maximum cross-sectional area of the organoid on day n to the total maximum cross-sectional area of the organoid on day 0.
根据本发明的实施例，类器官药物药效评价周期为15天。According to the embodiment of the present invention, the drug efficacy evaluation period of the organoid drug is 15 days.
根据本发明的实施例，所述药效评价周期内从第0天开始每隔3天对类器官进行所述断层图像拍摄。根据本发明实施例，对待测药物进行15天类器官面积标准化值监测，其中，类器官面积标准化值每隔3天检测一次，得到的肠癌类药物药效结果与病人用药后的临床反应具有很高的一致性。According to an embodiment of the present invention, the tomographic images of the organoids are captured every 3 days starting from the 0th day in the drug efficacy evaluation period. According to the embodiment of the present invention, the drug to be tested is monitored for 15 days of normalized value of the organoid area, wherein the normalized value of the organoid area is detected once every 3 days, and the obtained results of the drug efficacy of the intestinal cancer drug are consistent with the clinical response of the patient after medication. Very high consistency.
根据本发明的实施例，所述类器官为肿瘤类器官。According to an embodiment of the present invention, the organoid is a tumor organoid.
根据本发明的实施例，所述肿瘤类器官为肠癌类器官。According to an embodiment of the present invention, the tumor organoid is an intestinal cancer organoid.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:
图1是根据本发明的实施例的水凝胶类器官单平面图像分析法测定肿瘤药物药效结果图；将类器官培养在微腔包被的培养皿中，控制每个孔仅存在一个类器官。加入0.001～20μM的Akt抑制剂Afuresertib进行处理，拍摄单一截面统计每个类器官截面积大小，其中，右侧图横坐标为加药的种类及对应的浓度，加药的种类从左到右分别为：DMSO(二甲基亚砜)、Afuresertib(Akt抑制剂)、Gambogic acid(藤黄酸)，纵坐标为类器官面积；Fig. 1 is a graph showing the results of the determination of the efficacy of tumor drugs by the single-plane image analysis method of hydrogel organoids according to an embodiment of the present invention; the organoids are cultured in microcavity-coated petri dishes, and only one organoid exists in each well. organ. Add 0.001-20 μM Akt inhibitor Afuresertib for treatment, and take a single cross-section to count the cross-sectional area of each organoid. The abscissa on the right side is the type of drug added and the corresponding concentration, and the type of drug added is from left to right. For: DMSO (dimethyl sulfoxide), Afuresertib (Akt inhibitor), Gambogic acid (gambogic acid), the ordinate is the organoid area;
图2是根据本发明实施例的肠癌类器官培养流程图；将肿瘤患者手术或活检的肿瘤组织，消化为细胞悬液，培养在基质胶内，生长成为具有三维结构的肿瘤类器官；Fig. 2 is a flow chart of intestinal cancer organoid culture according to an embodiment of the present invention; the tumor tissue of a tumor patient undergoing surgery or biopsy is digested into a cell suspension, cultured in matrigel, and grown into a tumor organoid with a three-dimensional structure;
图3是根据本发明实施例的肠癌类器官对肠癌药物进行药物实验的实验设计图；将类器官种植在48孔板中，待直径达到100μm时分别加入5-氟尿嘧啶和开普拓进行处理，每隔三天更换一次培养基，培养周期为15天；Fig. 3 is an experimental design diagram of intestinal cancer organoids for drug experiments on intestinal cancer drugs according to an embodiment of the present invention; the organoids are planted in a 48-well plate, and when the diameter reaches 100 μm, 5-fluorouracil and captopor are respectively added to carry out the experiment. Treatment, the culture medium was replaced every three days, and the culture period was 15 days;
图4是根据本发明实施例的肠癌类器官对肠癌药物进行药物实验的过程中断层拍照的流程图及生成的叠加图像示例图；Fig. 4 is a flow chart of tomographic photography and an example diagram of superimposed images generated in the process of performing drug experiments on intestinal cancer drugs by intestinal cancer organoids according to an embodiment of the present invention;
图5是根据本发明实施例的肠癌类器官采用倒置金相显微镜连续断层扫描，总最大截面积随Z轴拍摄层间高度变化的趋势图；Fig. 5 is a continuous tomographic scan of intestinal cancer organoids using an inverted metallographic microscope according to an embodiment of the present invention, and a trend diagram of the total maximum cross-sectional area changing with the interlayer height taken along the Z axis;
图6是根据本发明实施例的肠癌类器官采用倒置金相显微镜连续断层扫描，Z轴拍摄层间高度为50μm(左)与150μm(右)截面积统计圈选结果的对比图；使用ImageJ进行圈选，150μm相较50μm丢失很多小型类器官的信号；Fig. 6 is a comparison diagram of the results of the statistical circles of the cross-sectional area of the intestinal cancer organoids according to the embodiment of the present invention using an inverted metallographic microscope for continuous tomographic scanning, and the z-axis shooting interlayer heights of 50 μm (left) and 150 μm (right); using ImageJ Carrying out circle selection, 150 μm loses many signals of small organoids compared with 50 μm;
图7是根据本发明实施例的肠癌类器官采用倒置金相显微镜连续断层扫描，更改光路前 (左)、后(右)观察到的类器官断层图像对比图；Fig. 7 is a comparison diagram of the tomographic images of intestinal cancer organoids observed before (left) and after (right) after changing the optical path by using an inverted metallographic microscope for continuous tomographic scanning of intestinal cancer organoids according to an embodiment of the present invention;
图8是根据本发明实施例的肠癌类器官采用倒置金相显微镜连续断层扫描，高曝光度(左)及低曝光度(右)条件下观察到的类器官断层图像的对比图；8 is a comparison diagram of the tomographic images of intestinal cancer organoids observed under high exposure (left) and low exposure (right) conditions using an inverted metallographic microscope for continuous tomographic scanning of intestinal cancer organoids according to an embodiment of the present invention;
图9是根据本发明实施例的肠癌类器官采用荧光显微镜连续断层扫描，总最大截面积随Z轴拍摄层间高度变化的趋势图；FIG. 9 is a trend diagram of the total maximum cross-sectional area of the colorectal cancer organoid according to an embodiment of the present invention using a fluorescence microscope for continuous tomographic scanning as the interlayer height changes along the Z-axis;
图10是根据本发明实施例的肠癌类器官采用倒置金相显微镜单层扫描获得的，第0天(左)及第6天(右)单焦平面类器官拍摄图；Fig. 10 is a single focal plane photograph of intestinal cancer organoids on the 0th day (left) and 6th day (right) obtained by single-layer scanning with an inverted metallographic microscope according to an embodiment of the present invention;
图11是根据本发明实施例的肠癌类器官采用倒置金相显微镜连续断层扫描获得的，第0天(左)及第6天(右)多张Z-Stack焦平面叠加图；Fig. 11 is the overlay of multiple Z-Stack focal planes on the 0th day (left) and the 6th day (right) obtained by continuous tomographic scanning of intestinal cancer organoids according to an embodiment of the present invention using an inverted metallographic microscope;
图12是根据本发明实施例的面积法采用倒置金相显微镜对肠癌类器官连续断层扫描获得的，5-氟尿嘧啶和开普拓处理后类器官总最大截面积趋势图；Fig. 12 is a trend diagram of the total maximum cross-sectional area of organoids after treatment with 5-fluorouracil and Capto, obtained by continuous tomographic scanning of intestinal cancer organoids using an inverted metallographic microscope by planimetric method according to an embodiment of the present invention;
图13是根据本发明实施例的面积法采用荧光显微镜对肠癌类器官连续断层扫描获得的荧光标记断层叠加图像，其中，虚线框圈出的为代表性的大小形态各异的单个类器官；Fig. 13 is a tomographic overlay image of fluorescent labeling obtained by sequential tomographic scanning of intestinal cancer organoids using a fluorescence microscope using the planimetric method according to an embodiment of the present invention, where the dotted line circles represent a single organoid of various sizes and shapes;
图14是根据本发明实施例的面积法采用荧光显微镜对肠癌类器官连续断层扫描获得的，5-氟尿嘧啶和开普拓处理后类器官总最大截面积趋势图；Fig. 14 is a trend diagram of the total maximum cross-sectional area of organoids after treatment with 5-fluorouracil and Capto, obtained by sequential tomographic scanning of intestinal cancer organoids using a fluorescence microscope using the planimetric method according to an embodiment of the present invention;
图15是根据本发明实施例的三维建模法采用高倍显微镜对肠癌类器官连续断层扫描获得的荧光标记断层图像；拍摄区间内共获得连续的56张图像，可以清楚的观察到类器官立体形态的变化，由最初无荧光信号，到渐渐拍摄到类器官上半部，中心部，下半部至信号丢失；Fig. 15 is a fluorescent-labeled tomographic image obtained by using a high-power microscope for continuous tomographic scanning of intestinal cancer organoids using a three-dimensional modeling method according to an embodiment of the present invention; a total of 56 consecutive images were obtained in the shooting interval, and the three-dimensional organoids can be clearly observed Morphological changes, from no fluorescent signal at the beginning, to gradually photographing the upper half, center, and lower half of the organoid until the signal is lost;
图16是根据本发明实施例的三维建模法采用高倍显微镜对肠癌类器官连续断层扫描获得的荧光标记断层图像采用MATLAB进行三维建模所得的示例图；左上为X轴方向横截面图，左下为Z轴方向最大截面所在的铅垂面图，右上为Y轴方向纵截面图，右下为Z轴方向最大截面所在的铅垂面类器官圈选结果，虚线框圈出的即为每一个形态和大小不一的类器官；Fig. 16 is an example diagram obtained by using a high-power microscope to perform three-dimensional modeling on fluorescently labeled tomographic images obtained by continuous tomographic scanning of intestinal cancer organoids using a high-power microscope according to the three-dimensional modeling method of an embodiment of the present invention; the upper left is a cross-sectional view in the X-axis direction, The lower left is the vertical plane view where the largest section in the Z-axis direction is located, the upper right is the longitudinal section view in the Y-axis direction, and the lower right is the result of organoid circle selection on the vertical plane where the largest cross-section in the Z-axis direction is located. An organoid of various shapes and sizes;
图17是根据本发明实施例的三维建模法采用高倍显微镜对肠癌类器官连续断层扫描获得的，5-氟尿嘧啶和开普拓处理后类器官总表面积趋势图；以及Fig. 17 is a three-dimensional modeling method according to an embodiment of the present invention obtained by serial tomographic scanning of intestinal cancer organoids using a high-power microscope, and a trend diagram of the total surface area of the organoids after treatment with 5-fluorouracil and Capto; and
图18是根据本发明实施例的肠癌类器官面积法对药物5-氟尿嘧啶和开普拓药效评价结果与病人临床用药后的反应结果对比图。类器官药效结果分为敏感和耐受(注：有些病人类器官依据其临床用药种类并未使用开普拓处理，即临床上病人也未使用开普拓)。临床病人的用药结果分为反应良好和反应差两类。图中可以看出，临床反应良好的病人，大多类器官药效为敏感，反应差的病人，类器官药效结果多数为耐受。面积法测药效与临床结果相比具有很高的一致性。敏感性为78.01％，特异性为91.97％，准确率为84.43％。Fig. 18 is a comparison chart of the evaluation results of the drug efficacy of the drugs 5-fluorouracil and Capto according to the colorectal cancer organoid area method according to the embodiment of the present invention and the response results of patients after clinical medication. Organoid drug efficacy results are divided into sensitive and tolerant (Note: Some human organs of patients have not been treated with Capto according to their clinical drug type, that is, patients have not been treated with Capto in clinical practice). The drug results of clinical patients are divided into two categories: good response and poor response. It can be seen from the figure that for patients with good clinical response, most of the organoid drug effects are sensitive, and for patients with poor response, most of the organoid drug effect results are tolerant. Compared with the clinical results, the drug efficacy measured by the planimetric method has a high consistency. The sensitivity was 78.01%, the specificity was 91.97%, and the accuracy was 84.43%.
发明详细描述Detailed description of the invention
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。Embodiments of the present invention are described in detail below. The embodiments described below are exemplary only for explaining the present invention and should not be construed as limiting the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。In addition, the terms "first" and "second" are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.
术语“任选地”仅用于描述目的，而不能理解为指示或暗示相对重要性。由此，限定有“任选地”的特征可以明示或者隐含地包括或不包括该特征。The term "optionally" is used for descriptive purposes only and is not to be understood as indicating or implying relative importance. Thus, a feature defined as "optionally" may be expressly or implicitly included or excluded.
以下实施例以肠癌类器官为模型，利用先进的显微摄影技术，精确的判定在加入肿瘤药物后，基质胶中类器官在整个用药周期过程中的大小变化，从而判断肿瘤药物药效，指导临床用药。In the following examples, intestinal cancer organoids are used as models, and advanced photomicrographing techniques are used to accurately determine the size change of organoids in Matrigel during the entire medication cycle after the addition of tumor drugs, thereby judging the efficacy of tumor drugs. guide clinical medication.
在一些实施方案中，发明人用倒置倒置金相显微镜进行扫描或钙黄绿素对类器官染色的条件下用倒置荧光显微镜进行扫描。只有在类器官形态良好时，倒置金相显微镜拍摄的类器官才能较好的区分类器官的生存和死亡情况，得较好的统计结果。当病人来源的类器官因个体差异导致形态各异，倒置金相显微镜拍摄下类器官的形态很难判断死活时，可以选择用钙黄绿素对类器官进行荧光标记，钙黄绿素仅标记活细胞，利用荧光显微镜进行类器官最大截面积的检测。所用倒置荧光显微镜进行断层扫描时激发光为紫外光；所用紫外光的波长为488nm。In some embodiments, the inventors scan with an inverted metallographic microscope or scan with an inverted fluorescence microscope under conditions where the organoids are stained with calcein. Only when the organoids are in good shape, the organoids photographed by the inverted metallographic microscope can better distinguish the survival and death of the organoids, and obtain better statistical results. When organoids derived from patients have different shapes due to individual differences, and it is difficult to determine whether the organoids are alive or dead by the morphology of the organoids taken by an inverted metallographic microscope, you can choose to use calcein to fluorescently label the organoids. Calcein only labels living cells. Fluorescence microscopy was used to detect the maximum cross-sectional area of organoids. When the inverted fluorescent microscope is used for tomographic scanning, the excitation light is ultraviolet light; the wavelength of the ultraviolet light used is 488nm.
在一些实施方案中，每个处理组中有3个重复培养孔，每个培养孔中有多个类器官，对于可以利用倒置金相显微镜进行可见光成像的形态良好的类器官，可以直接利用倒置金相显微镜进行断层扫描；当病人来源的类器官因个体差异导致形态各异，倒置金相显微镜拍摄下类器官的形态很难判断死活时，可以选择用钙黄绿素对类器官进行荧光标记，钙黄绿素仅标记活细胞，利用荧光显微镜进行类器官最大截面积的检测。In some embodiments, there are three replicate wells per treatment group with multiple organoids in each well, and for well-formed organoids that can be imaged with visible light using an inverted metallographic microscope, the inverted metallurgical microscope can be used directly. Metallographic microscope for tomographic scanning; when the organoids derived from patients have different shapes due to individual differences, and it is difficult to judge whether the organoids are alive or dead by the morphology of the organoids taken by an inverted metallographic microscope, you can choose to fluorescently label the organoids with calcein. Chlorophyll only labels living cells, and the maximum cross-sectional area of organoids is detected using a fluorescence microscope.
在一些实施方案中，将每个培养孔中的断层图像生成一张叠加图像，利用叠加图像可以精确的找出基质胶中每一个类器官的位置和最大截面积，并获得单个培养孔中所有类器官最大截面积之和，再将同一个处理组的3个重复孔获得的3个最大截面积再次加和并取平均值，在本发明中称该平均值为类器官的总最大截面积。依据类器官的最大截面积计算出类器官的总最大截面积，依据类器官的总最大截面积计算出类器官的总最大截面积的标准 化值，从而评价类器官大小，类器官的大小即可反映药物的药效。该方法排除了类器官在基质胶中的位置，分布，大小，形态和异质性差异带来的统计误差；而且在整个加药周期中，可以在保持类器官原有状态的情况下，选取不同时间节点进行统计，观察不同时间点类器官的大小差异，从而精确的判定加药后的药效，为临床诊断和用药提供依据。经临床药效一致性验证证明，所述方法所测得药物药效评价结果与临床药效评价结果一致，因此，该方法是目前最准确的肿瘤类器官药物药效判定方法。In some embodiments, an overlay image is generated from the tomographic image in each culture well, and the position and maximum cross-sectional area of each organoid in Matrigel can be precisely found out by using the overlay image, and all the organs in a single culture well can be obtained. The sum of the maximum cross-sectional area of the organoid, and then sum the three maximum cross-sectional areas obtained from the three repeated wells of the same treatment group and take the average value, which is called the total maximum cross-sectional area of the organoid in the present invention . Calculate the total maximum cross-sectional area of the organoid based on the maximum cross-sectional area of the organoid, and calculate the normalized value of the total maximum cross-sectional area of the organoid based on the maximum total cross-sectional area of the organoid, so as to evaluate the size of the organoid, the size of the organoid is sufficient reflect the efficacy of the drug. This method eliminates statistical errors caused by differences in the position, distribution, size, shape and heterogeneity of organoids in Matrigel; Statistics are carried out at different time points, and the size difference of organoids at different time points is observed, so as to accurately determine the drug effect after drug addition, and provide a basis for clinical diagnosis and drug use. The consistency verification of clinical drug efficacy proves that the drug efficacy evaluation results measured by the method are consistent with the clinical drug efficacy evaluation results. Therefore, this method is currently the most accurate method for judging drug efficacy of tumor organoids.
下面将结合实施例对本发明的方案进行解释。The solutions of the present invention will be explained below in conjunction with examples.
实施例1肠癌类器官培养Example 1 Colon cancer organoid culture
将收集到的新鲜肠癌活检肿瘤样本拍照并记录具体信息，比对无误后用预冷的PBS(加入100×penicillin/streptomycin)洗5次，每次5min。在无菌操作台上，将无菌培养皿置于冰上，并倒入适量预冷灭菌的PBS，然后将癌组织置于其中，用无菌刀片将肿瘤样本切成细小碎片后移入8mL经37℃预热的消化液中。消化液配方为：7mL DMEM培养基(GIBCO,C1199500BT)，500U/mL胶原酶IV(Sigma-aldrich,C9407)，1.5mg/mL胶原酶II(Solarbio,C8150)、20μg/mL透明质酸酶(Solarbio,h8030)、0.1mg/mL中性蛋白酶II(Sigma-aldrich,D4693)、10μM RHOK抑制剂ly27632(Sigma-aldrich,Y0503)和1％胎牛血清。37℃水浴摇床消化40min。消化完毕后200g离心5min，用预冷的灭菌PBS洗5次，每次5min。然后200g离心沉淀所有细胞团(离心前取20μL镜下观察并计算细胞数量)。提取后用预冷1mL枪头加入适量基质胶(康宁，#356231)，使每50μL基质胶中细胞团数量为200个左右，随后用200μL预冷枪头悬浮混匀，接种于预热24孔板中，每孔50μL。接种后培养板置于37℃恒温培养箱中孵育5-8min后拿出。每孔加入500μL类器官培养基(丹望医疗，Cat#K20001)，镜下观察接种情况，随后置于培养箱中培养。每日观察类器官生长情况并用显微镜进行拍照记录，每3日更换一次培养基，整体流程如图2所示。The collected fresh colon cancer biopsy tumor samples were photographed and the specific information was recorded, and after comparison, they were washed 5 times with pre-cooled PBS (adding 100× penicillin/streptomycin), 5 min each time. On the aseptic operating table, put the sterile petri dish on ice, pour an appropriate amount of pre-cooled and sterilized PBS, and then place the cancer tissue in it, cut the tumor sample into small pieces with a sterile blade, and transfer it into 8mL Digestion solution preheated at 37°C. The digestive solution formula is: 7mL DMEM medium (GIBCO, C1199500BT), 500U/mL collagenase IV (Sigma-aldrich, C9407), 1.5mg/mL collagenase II (Solarbio, C8150), 20μg/mL hyaluronidase ( Solarbio, h8030), 0.1 mg/mL dispase II (Sigma-aldrich, D4693), 10 μM RHOK inhibitor ly27632 (Sigma-aldrich, Y0503) and 1% fetal bovine serum. Digest for 40 minutes in a water bath shaker at 37°C. After digestion, centrifuge at 200g for 5min, wash with pre-cooled sterilized PBS 5 times, 5min each time. Then centrifuge at 200g to pellet all cell clusters (take 20 μL before centrifugation to observe under the microscope and count the number of cells). After extraction, add an appropriate amount of Matrigel (Corning, #356231) with a pre-cooled 1mL pipette tip, so that the number of cell clusters in each 50μL Matrigel is about 200, then suspend and mix with a 200μL pre-cooled pipette tip, and inoculate in a preheated 24-well plate 50 μL per well. After inoculation, the culture plate was incubated in a constant temperature incubator at 37°C for 5-8 minutes and then taken out. Add 500 μL of organoid culture medium (Danwang Medical, Cat#K20001) to each well, observe the inoculation situation under a microscope, and then culture in an incubator. Observe the growth of organoids every day and record them with a microscope, and replace the medium every 3 days. The overall process is shown in Figure 2.
1-2周传代一次。传代时，将培养孔内培养基吸去，加入500mL预冷PBS将培养板置于冰盒上放置片刻，预冷枪头将基质胶吹散，移入15mL无菌离心管中吹打将基质胶完全吹散混匀，4℃离心机69g离心5min，将上清及沉淀在底部的基质胶吸去。再加入预冷PBS重复洗三次即可除去基质胶。加入5mL预冷PBS重新悬浮管底类器官，吹打50～100次将类器官吹打成细胞团(镜下观察细胞团已被吹打成单细胞或细胞团即可)，4℃离心机69g离心5min。按类器官培养流程继续培养，传代时按每孔1比4的比例接种，培养基成分如表1所示。Subculture once every 1-2 weeks. When subculture, suck out the medium in the culture well, add 500mL pre-cooled PBS, place the culture plate on the ice box for a while, blow off the matrigel with the pre-cooled pipette tip, transfer it into a 15mL sterile centrifuge tube and blow the matrigel completely Disperse and mix well, centrifuge at 69g at 4°C for 5min, and suck off the supernatant and Matrigel deposited at the bottom. Then add pre-cooled PBS and repeat washing three times to remove Matrigel. Add 5mL pre-cooled PBS to resuspend the organoids at the bottom of the tube, pipette 50-100 times to pipette the organoids into cell clusters (observe under the microscope that the cell clusters have been blown into single cells or cell clusters), centrifuge at 4°C for 69g Centrifuge for 5min. Continue to culture according to the organoid culture process, inoculate at a ratio of 1 to 4 per well when subcultured, and the medium composition is shown in Table 1.
表1：肠癌类器官培养基组分表Table 1: Components of Intestinal Cancer Organoid Medium
| 组分名称component name | 公司company | 货号Item No. | 母液浓度Mother liquor concentration | 溶剂solvent | 终浓度Final concentration |
| Advanced DMEM/F12Advanced DMEM/F12 | GIBCOGIBCO | 12634-01012634-010 | -- | 1×1× | 1×1× |
|
R-spondin 1R- |
Sino Biological Inc.Sino Biological Inc. | 11083-HNAS11083-HNAS | 50mg/mL50mg/mL | 0.1％BSA/PBS0.1%BSA/PBS | 500ng/mL500ng/mL |
| NogginNoggin | Sino Biological Inc.Sino Biological Inc. | 50688-M02H50688-M02H | 10mg/mL10mg/mL | 0.1％BSA/PBS0.1%BSA/PBS | 100ng/mL100ng/mL |
| EGFEGF | Sino Biological Inc.Sino Biological Inc. | 50482-MNCH50482-MNCH | 500mg/mL500mg/mL | 0.1％BSA/PBS0.1%BSA/PBS | 50ng/mL50ng/mL |
| HEPESHEPES | GIBCOGIBCO | 1563008015630080 | 100×100× | -- | 1×1× |
| GlutamaxGlutamax | GIBCOGIBCO | 3505006135050061 | 100×100× | -- | 1×1× |
| NormocinNormocin | InvivoGenInvivoGen | ant-nr-1ant-nr-1 | 500×500× | -- | 1×1× |
| Gentamicin/amphoteritin BGentamicin/amphoteritin B | GIBCOGIBCO | R01510R01510 | 500×500× | -- | 1×1× |
| N2N2 | InvitrogenInvitrogen | 17502-04817502-048 | 50×50× | -- | 1×1× |
| B27B27 | InvitrogenInvitrogen | 17504-04417504-044 | 100×100× | -- | 1×1× |
| n-Acetylcysteinen-Acetylcysteine | Sigma-aldrichSigma-aldrich | A9165A9165 | 500mM500mM | ddH 2O ddH 2 O | 1mM1mM |
| NiacinamideNiacinamide | Sigma-aldrichSigma-aldrich | N0636N0636 | 1M1M | ddH 2O ddH 2 O | 10mM10mM |
| A-83-01A-83-01 | TocrisTocris | 29392939 | 5mM5mM | DMSODMSO | 500nM500nM |
| SB202190SB202190 | Sigma-aldrichSigma-aldrich | S7067S7067 | 30M30M | DMSODMSO | 3μM3μM |
| GastrinGastrin | Sigma-aldrichSigma-aldrich | G9145G9145 | 100μM100μM | 0.1％BSA/PBS0.1%BSA/PBS | 10nM10nM |
| Prostaglandin E2Prostaglandin E2 | Sigma-aldrichSigma-aldrich | P6532P6532 | 100μM100μM | DMSODMSO | 10nM10nM |
实施例2倒置金相显微镜拍摄类器官参数优化Example 2 Optimization of Organoid Parameters Shooting with an Inverted Metallographic Microscope
选取培养状态良好的类器官，按实施例1的方法消化并传代至48孔板中。初始接种类器官密度为10-15/μL基质胶，每孔加入300μL培养基。最终每个孔10μL基质胶中含有200±50个类器官，当类器官直径达到100μm时进行参数优化实验，共设置3个重复孔，拍摄周期为15天，从第0天开始每隔3天对每个孔的类器官进行光学成像，用Capture 2.1拍摄软件的EDF景深扩展功能结合倒置金相显微镜，进行递进式断层扫描输出多张Z-Stack图。拍摄软件会依据每层吸光值，运用小波算法将多张Z-Stack图进行叠加，生成一张完整的包含所有拍摄到的类器官的叠加图像，具体操作流程如图4所示。Organoids with good culture status were selected, digested and passaged into 48-well plates according to the method of Example 1. The initial inoculation density of organoids was 10-15/μL Matrigel, and 300 μL medium was added to each well. Finally, 200±50 organoids were contained in 10 μL Matrigel in each well. When the diameter of the organoid reached 100 μm, the parameter optimization experiment was performed. A total of 3 replicate wells were set up. The shooting cycle was 15 days, starting from day 0 every 3 days Perform optical imaging of the organoids in each well, and use the EDF depth-of-field expansion function of the Capture 2.1 shooting software combined with an inverted metallographic microscope to perform progressive tomographic scanning and output multiple Z-Stack images. The shooting software will use the wavelet algorithm to superimpose multiple Z-Stack images based on the light absorption value of each layer to generate a complete superimposed image containing all the captured organoids. The specific operation process is shown in Figure 4.
1、拍摄区间优化1. Optimize the shooting range
由于基质胶与细胞不同为半球状固体，因此需要先确定基质胶的拍摄顶部和底部区间，即从哪里开始拍，到哪里结束。经过对该参数的优化，发明人得出10μL半球状基质胶的拍摄区间约为1500μm较佳。Since matrigel is a hemispherical solid different from cells, it is necessary to determine the top and bottom intervals of matrigel shooting, that is, where to start shooting and where to end. After optimizing the parameters, the inventors concluded that the imaging range of 10 μL of hemispherical matrigel is about 1500 μm, which is better.
2、拍摄层间高度优化2. Optimize the height between shooting layers
层间高度，即断层拍摄时，在拍摄区间内选择每隔多少微米拍一张照片才会使最终输出的图像最佳。发明人设置不同Z轴的拍摄层高为10、25、50、75、100、150μm，具体结果如图5、6所示。由图5可知，随着Z轴拍摄层间高度的增大，所得类器官总截面积越来越小，且图6所示的面积统计圈选可以看出层间高度150μm与50μm相比，丢失了很多小的类器官信号；10、25、50μm面积统计结果基本无差异；此外，层间高度设置越小，耗时越长，计算机及拍摄机器耗损越大。因此，综合上述结果，发明人选择50μm作为层间高度的设置值。Inter-layer height, that is, when tomography is taken, how many microns are selected in the shooting interval to take a photo to make the final output image the best. The inventors set the shooting layer heights of different Z axes as 10, 25, 50, 75, 100, and 150 μm, and the specific results are shown in Figures 5 and 6. It can be seen from Figure 5 that the total cross-sectional area of the obtained organoids is getting smaller and smaller as the interlayer height of the Z-axis photography increases, and the area statistical circle shown in Figure 6 shows that the interlayer height of 150 μm is compared with that of 50 μm. Many small organoid signals are lost; there is basically no difference in the statistical results of 10, 25, and 50 μm areas; in addition, the smaller the interlayer height is set, the longer it takes, and the greater the consumption of computers and shooting machines. Therefore, based on the above results, the inventors selected 50 μm as the set value of the interlayer height.
3、光路和曝光度3. Light path and exposure
由于目前市面上的高内涵仪器在拍摄培养类器官或细胞的多孔板(平底板)时，聚光镜发散出平行光α，在孔板内部，形成光折射，产生β夹角，造成光照不均匀现象；使靠近孔板边缘的类器官，无法清晰观察。因此，发明人对光路进行改变，在所用48孔板顶部加入匀光板，打乱平行光路，使其成为均匀的散射光，如图7所示。为了输出准确的统计图像，还需要确定最佳曝光度，以统一叠加时使用的光强度，过度曝光或曝光太低都会导致拍出来的图像效果不佳，干扰统计结果。经过筛选，发明人发现，将LUT(look up table)设置在50-55k范围内效果最佳，所得图像如图8所示。When the current high-content instruments on the market shoot the porous plate (flat-bottomed plate) for culturing organoids or cells, the condenser lens emits parallel light α, which forms light refraction inside the well plate and produces an included angle of β, resulting in uneven illumination. ; so that organoids near the edge of the well plate cannot be clearly observed. Therefore, the inventors changed the optical path by adding a uniform light plate on the top of the 48-well plate used to disrupt the parallel optical path and make it a uniform scattered light, as shown in FIG. 7 . In order to output accurate statistical images, it is also necessary to determine the optimal exposure to unify the light intensity used during superimposition. Overexposure or too low exposure will result in poor image quality and interfere with statistical results. After screening, the inventor found that setting the LUT (look up table) in the range of 50-55k works best, and the resulting image is shown in Figure 8.
实施例3荧光显微镜拍摄类器官层间高度参数优化Example 3 Parameter optimization of interlayer height of organoids photographed by fluorescence microscope
选取培养状态良好的类器官，按实施例1的方法消化并传代至48孔板中。初始接种类器官密度为10-15/μL基质胶，每孔加入300μL培养基。最终每个孔10μL基质胶中含有200±50个类器官，当类器官直径达到100μm时进行参数优化实验，共设置3个重复孔，拍摄周期为15天，从第0天开始每隔3天对每个孔的类器官进行光学成像，用Capture 2.1拍摄软件的EDF景深扩展功能结合倒置荧光显微镜，进行递进式断层扫描输出多张Z-Stack图。拍摄软件会依据每层吸光值，运用小波算法将多张Z-Stack图进行叠加，生成一张完整的包含所有拍摄到的类器官的叠加图像，具体操作流程如图4所示。Organoids with good culture status were selected, digested and passaged into 48-well plates according to the method of Example 1. The initial inoculation density of organoids was 10-15/μL Matrigel, and 300 μL medium was added to each well. Finally, 200±50 organoids were contained in 10 μL Matrigel in each well. When the diameter of the organoid reached 100 μm, the parameter optimization experiment was performed. A total of 3 replicate wells were set up. The shooting cycle was 15 days, starting from day 0 every 3 days Perform optical imaging of the organoids in each well, and use the EDF depth-of-field expansion function of the Capture 2.1 shooting software combined with an inverted fluorescence microscope to perform progressive tomographic scanning and output multiple Z-Stack images. The shooting software will use the wavelet algorithm to superimpose multiple Z-Stack images based on the light absorption value of each layer to generate a complete superimposed image containing all the captured organoids. The specific operation process is shown in Figure 4.
发明人采用钙黄绿素对类器官进行荧光标记，钙黄绿素仅标记活细胞。在培养周期的15天里，从第0天开始每隔3天对每个孔的多个类器官进行钙黄绿素标记和荧光成像。在培养基中加入终浓度4μM的钙黄绿素染色60min，用Capture 2.1拍摄软件的EDF景深扩展功能结合倒置荧光显微镜，用488nm激发光显现绿色荧光图片。发明人对Z轴的拍摄层间高度进行筛选，将层间高度设置为25、50、100、150、200μm，结果表明10μL半球状基质胶的拍摄区间约为100μm较佳，其余参数与实施例2一致，具体结果如图9所示。The inventors used calcein to fluorescently label organoids, and calcein only labels living cells. Multiple organoids per well were labeled with calcein and fluorescently imaged every 3 days starting at day 0 during the 15-day culture period. Calcein with a final concentration of 4 μM was added to the medium for staining for 60 min, and the EDF depth-of-field expansion function of Capture 2.1 shooting software combined with an inverted fluorescence microscope was used to display green fluorescence pictures with 488 nm excitation light. The inventor screened the interlayer height of the Z-axis, and set the interlayer height to 25, 50, 100, 150, and 200 μm. The results showed that the imaging interval of 10 μL hemispherical matrigel was about 100 μm, and the remaining parameters were the same as those in the examples 2, the specific results are shown in Figure 9.
实施例4连续断层拍摄与传统单层拍摄图像对比Example 4 Contrast between continuous tomographic imaging and traditional single-layer imaging
将实施例2种获得的类器官用倒置金相显微镜分别获得传统单视野图像，及根据实施例2中的参数进行断层扫描获得的叠加图像。单层拍摄得到的图片经常包含很多虚焦面类器官，既无法观测类器官存活情况，也为面积统计带来困难，往往导致实验结果误差偏大。在第0天时，寻找到一个大多数类器官都在同拍摄焦面内的视野，使用单焦平面拍摄；在第6天时，与第0天同视野内，类器官经过培养增大，焦平面变得不统一，如图10所示，不能被聚焦的类器官，在成像时，形成光斑虚影，导致可观察到的类器官数量丢失，形成误差。连续断层拍摄很好的避免了上述弊端，生成的图像类器官清晰，面积统计便捷。在第0天时，寻找到一个，大多数类器官都在同拍摄焦面内的视野，使用Z-stack多层焦平面拍摄，并使用EDF功能合成为一张叠加图像。第6天时采用同样的方法拍摄，能较好的在一张叠加图像上观察到不同焦平面的多个类器官，如图11所示。The organoids obtained in Example 2 were used to obtain traditional single-view images and superimposed images obtained by tomographic scanning according to the parameters in Example 2 using an inverted metallographic microscope. The pictures obtained by single-layer shooting often contain many virtual focus organoids, which can not only observe the survival of organoids, but also bring difficulties to area statistics, which often leads to large errors in experimental results. On day 0, a field of view where most of the organoids were in the same focal plane was found, and a single focal plane was used for shooting; on day 6, in the same field of view as day 0, the organoids were cultured and enlarged, and the focal plane Become inconsistent, as shown in Figure 10, organoids that cannot be focused will form ghost spots during imaging, resulting in the loss of the number of observable organoids, resulting in errors. Continuous tomographic photography avoids the above-mentioned disadvantages well, and the generated images of organoids are clear, and the area statistics are convenient. On day 0, we found a field of view in which most organoids were in the same focal plane, and used the Z-stack multi-layer focal plane to shoot, and used the EDF function to synthesize a superimposed image. The same method was used to shoot on the 6th day, and multiple organoids with different focal planes could be well observed on one superimposed image, as shown in Figure 11.
实施例5面积法评价药物药效准确性验证Embodiment 5 area method evaluates drug efficacy accuracy verification
选取培养状态良好的类器官，按实施例1的方法消化并传代至48孔板中。初始接种类器官密度为10-15/μL基质胶，每孔加入300μL培养基。最终每个孔10μL基质胶中含有200±50个类器官，当类器官直径达到100μm时进行药物处理。2个实验组，包括5-氟尿嘧啶(5-Fu)组和开普拓(CPT-11)组。分别将实验组类器官的培养基中加入10mM 5-Fu(Selleck,S1209)或10mM CPT-11(Selleck,S2217)，3天后用加入药物的培养基更换旧培养基，后续用不含药物的培养基每3天更换一次。从进行加药时起，进行为期15天的培养，具体操作流程同实施例2，后续实验操作如下：Organoids with good culture status were selected, digested and passaged into 48-well plates according to the method of Example 1. The initial inoculation density of organoids was 10-15/μL Matrigel, and 300 μL medium was added to each well. Finally, 200 ± 50 organoids were contained in 10 μL of Matrigel per well, and drug treatment was performed when the diameter of organoids reached 100 μm. Two experimental groups, including 5-fluorouracil (5-Fu) group and Capto (CPT-11) group. Add 10mM 5-Fu (Selleck, S1209) or 10mM CPT-11 (Selleck, S2217) to the culture medium of the organoids in the experimental group respectively, replace the old medium with the medium added with the drug after 3 days, and then replace it with the medium without the drug The medium was changed every 3 days. From the time of dosing, carry out a period of 15 days of cultivation, the specific operation process is the same as in Example 2, and the subsequent experimental operations are as follows:
1、面积法评价药物药效1. Evaluation of drug efficacy by area method
1)倒置金相显微镜拍摄类器官1) Photographing organoids with an inverted metallographic microscope
本实验设置2个实验组，每个处理组设置3个重复孔。In this experiment, two experimental groups were set up, and three replicate wells were set up for each treatment group.
在加药后的15天里，从第0天开始每隔3天对两种药物5-Fu和CPT-11处理的每个孔的类器官进行光学成像，具体操作流程及参数设置同实施例2。In the 15 days after the addition of the drug, the organoids in each well treated with the two drugs 5-Fu and CPT-11 were optically imaged every 3 days from the 0th day, and the specific operation process and parameter settings were the same as in the example 2.
使用Image-Pro Plus 6.0(Media Cybernetics,Inc.)或Image J软件(National Institute of Health,USA)测量每个培养孔叠加图像中类器官个数及每个类器官最大截面积，将每个培养孔中多个类器官的最大截面积相加，再计算每个处理组中3个重复培养孔最大截面积加和后的平均值，获得该处理组的总最大截面积。将第0天的类器官总最大截面积标准化为100％，第1天及随后天数统计的类器官总最大截面积与第0天总最大截面积的比值为标准化值，即获得培养周期内每3天的类器官总最大截面积百分比，具体结果如表2所示。用GraphPad Prism制作不同处理方式下类器官加药前后总最大截面积变化，如图12所示。依 据加药后类器官总最大截面积的标准化值变化的结果推断肿瘤药物的药效，用于临床用药指导。Use Image-Pro Plus 6.0 (Media Cybernetics, Inc.) or Image J software (National Institute of Health, USA) to measure the number of organoids in the superimposed images of each culture well and the maximum cross-sectional area of each organoid, and divide each culture Add the maximum cross-sectional areas of multiple organoids in the well, and then calculate the average value of the sum of the maximum cross-sectional areas of the three repeated culture wells in each treatment group to obtain the total maximum cross-sectional area of the treatment group. The total maximum cross-sectional area of organoids on day 0 was normalized to 100%, and the ratio of the total maximum cross-sectional area of organoids counted on day 1 and subsequent days to the total maximum cross-sectional area on day 0 was the standardized value, that is, to obtain The percentage of the total maximum cross-sectional area of organoids at day 3 is shown in Table 2. Use GraphPad Prism to make changes in the total maximum cross-sectional area of organoids before and after adding drugs under different treatment methods, as shown in Figure 12. According to the result of the change of the normalized value of the total maximum cross-sectional area of the organoid after drug addition, the drug effect of the tumor drug is inferred, which is used for clinical drug guidance.
表2：Table 2:
| 天数number of days | 5-氟尿嘧啶组5-fluorouracil group |
开普拓组 |
| 00 | 100±8.2100±8.2 | 100±7.5100±7.5 |
| 33 | 110±10.7110±10.7 | 147±8.9147±8.9 |
| 66 | 80±7.280±7.2 | 50±11.750±11.7 |
| 99 | 60±8.160±8.1 | 40±6.640±6.6 |
| 1212 | 48±5.048±5.0 | 20±4.820±4.8 |
| 1515 | 36±6.236±6.2 | 16±8.516±8.5 |
2)倒置荧光显微镜拍摄类器官2) Photographing organoids with an inverted fluorescence microscope
本实验设置2个实验组，每个处理组设置18个孔。In this experiment, two experimental groups were set up, and each treatment group set up 18 wells.
发明人采用钙黄绿素对类器官进行荧光标记，钙黄绿素仅标记活细胞。在加药后的15天里，从第0天开始每隔3天对每个处理的3个孔的类器官进行钙黄绿素标记和荧光成像。在每次检测的3个孔的培养基中加入终浓度4μM的钙黄绿素染色60min，染色后的类器官不再使用；用Capture 2.1拍摄软件的EDF景深扩展功能结合倒置荧光显微镜，用488nm激发光显现绿色荧光，具体操作流程及参数设置与实施例3一致，所得荧光标记的断层叠加图像如图13所示。The inventors used calcein to fluorescently label organoids, and calcein only labels living cells. Organoids from 3 wells per treatment were calcein-labeled and fluorescently imaged at 3-day intervals starting from day 0 for 15 days after dosing. Calcein with a final concentration of 4 μM was added to the culture medium of 3 wells for each detection and stained for 60 minutes, and the stained organoids were not used anymore; the EDF depth-of-field expansion function of the Capture 2.1 shooting software combined with an inverted fluorescence microscope was used to excite with 488nm Green fluorescence appeared, and the specific operation process and parameter settings were consistent with those in Example 3. The tomographic superimposed image of the obtained fluorescent marker is shown in FIG. 13 .
使用Image-Pro Plus 6.0(Media Cybernetics,Inc.)或Image J软件(National Institute of Health,USA)测量每个培养孔叠加图像中类器官个数及每个类器官最大截面积，将每个培养孔中多个类器官的最大截面积相加，再计算每个处理组中3个重复培养孔最大截面积加和后的平均值，获得该处理组的总最大截面积。将第0天的类器官总最大截面积标准化为100％，第1天及随后天数统计的类器官总最大截面积与第0天总最大截面积的比值为标准化值，即获得培养周期内每3天的类器官总最大截面积百分比，具体结果如表3所示。用GraphPad Prism制作不同处理方式下类器官加药前后总最大截面积变化，如图14所示。Use Image-Pro Plus 6.0 (Media Cybernetics, Inc.) or Image J software (National Institute of Health, USA) to measure the number of organoids in the superimposed images of each culture well and the maximum cross-sectional area of each organoid, and divide each culture Add the maximum cross-sectional areas of multiple organoids in the well, and then calculate the average value of the sum of the maximum cross-sectional areas of the three repeated culture wells in each treatment group to obtain the total maximum cross-sectional area of the treatment group. The total maximum cross-sectional area of organoids on day 0 was normalized to 100%, and the ratio of the total maximum cross-sectional area of organoids counted on day 1 and subsequent days to the total maximum cross-sectional area on day 0 was the standardized value, that is, to obtain The percentage of the total maximum cross-sectional area of organoids at day 3, the specific results are shown in Table 3. Use GraphPad Prism to make changes in the total maximum cross-sectional area of organoids before and after adding drugs under different treatment methods, as shown in Figure 14.
表3:table 3:
| 天数number of days | 5-氟尿嘧啶组5-fluorouracil group |
开普拓组 |
| 00 | 100±7.5100±7.5 | 100±6.5100±6.5 |
| 33 | 123±11.7123±11.7 | 167±6.8167±6.8 |
| 66 | 88±8.288±8.2 | 64±10.464±10.4 |
| 99 | 66±8.166±8.1 | 44±8.644±8.6 |
| 1212 | 51±6.451±6.4 | 31±5.731±5.7 |
| 1515 | 37±5.237±5.2 | 24±8.524±8.5 |
2、三维建模法评价药物药效2. Three-dimensional modeling method to evaluate drug efficacy
本实验设置2个实验组，每个处理组设置18个孔。In this experiment, two experimental groups were set up, and each treatment group set up 18 wells.
发明人采用钙黄绿素对类器官进行荧光标记，钙黄绿素仅标记活细胞。在加药后的15天里，从第0天开始每隔3天对每个处理的3个孔的类器官进行钙黄绿素标记和荧光成像。在每次检测的3个孔的培养基中加入终浓度4μM的钙黄绿素染色60min，染色后的类器官不再使用；利用荧光染色后的类器官结合40倍以上可过滤杂光的高倍显微镜对类器官进行断层拍摄，如图15所示。The inventors used calcein to fluorescently label organoids, and calcein only labels living cells. Organoids from 3 wells per treatment were calcein-labeled and fluorescently imaged at 3-day intervals starting from day 0 for 15 days after dosing. Calcein with a final concentration of 4 μM was added to the culture medium of 3 wells for each detection and stained for 60 minutes, and the stained organoids were not used again; the organoids after fluorescent staining were combined with a high-power microscope that could filter stray light more than 40 times to analyze The organoids were tomographically photographed, as shown in Figure 15.
采用MATLAB对获得的断层图像进行三维建模，如图16所示，利用三维建模统计加药后类器官的表面积。将第0天的类器官总表面积标准化为100％，随后天数统计的总表面积与第0天总表面积的比值为标准化值，获得培养周期内每隔3天类器官的总表面积百分比，具体结果如表4所示。用GraphPad Prism制作不同处理方式下类器官加药前后总面积标准化值的变化结果图，如图17所示。依据加药后类器官总表面积的标准化随时间变化的结果推断肿瘤药药效，用于临床用药指导。Using MATLAB to perform three-dimensional modeling on the obtained tomographic image, as shown in Figure 16, using three-dimensional modeling to count the surface area of the organoid after drug addition. The total surface area of the organoids on day 0 was normalized to 100%, and the ratio of the total surface area counted on subsequent days to the total surface area on day 0 was the normalized value to obtain the percentage of the total surface area of organoids every 3 days during the culture period. The specific results are as follows Table 4 shows. Use GraphPad Prism to make graphs of the change results of the normalized value of the total area of organoids before and after adding drugs under different treatment methods, as shown in Figure 17. According to the results of normalization of the total surface area of organoids over time after drug addition, the drug efficacy of tumor drugs can be inferred, which can be used for clinical drug guidance.
由上述三部分实验结果可以看出，采用面积法利用倒置金相显微镜或荧光标记后通过荧光显微镜连续断层扫描类器官叠加统计其最大截面变化以评价药物药效，与采用三维建模的方法统计类器官表面积变化以评价药物药效，两种方法所得到的药效评价结果一致。From the experimental results of the above three parts, it can be seen that using the planimetric method using an inverted metallographic microscope or fluorescent labeling to scan organoids through continuous tomographic scanning of the fluorescence microscope to overlay and count the maximum cross-sectional changes to evaluate the efficacy of drugs is different from the statistical method using three-dimensional modeling. The changes in the surface area of the organoids were used to evaluate the efficacy of the drug, and the results of the efficacy evaluation obtained by the two methods were consistent.
表4：Table 4:
| 天数number of days | 5-氟尿嘧啶组5-fluorouracil group |
开普拓组 |
| 00 | 100±10.2100±10.2 | 100±9.4100±9.4 |
| 33 | 177±12.7177±12.7 | 201±10.7201±10.7 |
| 66 | 145±8.3145±8.3 | 84±11.984±11.9 |
| 99 | 77±8.077±8.0 | 56±7.556±7.5 |
| 1212 | 62±7.162±7.1 | 35±5.835±5.8 |
| 1515 | 36±6.036±6.0 | 21±8.021±8.0 |
实施例6面积法测药效与临床药效一致性验证 Embodiment 6 area method measures drug effect and clinical drug effect consistency verification
发明人采用面积法测量80个肠癌患者来源的类器官对临床药物5-氟尿嘧啶和开普拓的敏感性，并与病人临床用药后的反应进行一致性验证。如图18所示，类器官药效结果分为敏感和耐受(注：有些病人类器官依据其临床用药种类并未使用开普拓处理，即临床上病人也未使用开普拓)，临床病人的用药结果分为反应良好和反应差两类；从图中可以看出，临床反应良好的病人，大多类器官药效为敏感，反应差的病人，类器官药效结果多数为耐 受。面积法测药效与临床结果相比具有很高的一致性。敏感性为78.01％，特异性为91.97％，准确率为84.43％。The inventors measured the sensitivity of 80 intestinal cancer patient-derived organoids to the clinical drugs 5-fluorouracil and captocin by using the planimetric method, and verified the consistency with the clinical drug responses of patients. As shown in Figure 18, the drug efficacy results of organoids are divided into sensitive and tolerant (Note: Some human organs of patients have not been treated with Capto according to their clinical drug type, that is, clinically patients have not used Capto). The drug results of patients are divided into two categories: good response and poor response; as can be seen from the figure, for patients with good clinical response, most of the organoid drug effects are sensitive, and for patients with poor response, most of the organoid drug effect results are tolerant. Compared with the clinical results, the drug efficacy measured by the planimetric method has a high consistency. The sensitivity was 78.01%, the specificity was 91.97%, and the accuracy was 84.43%.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the described specific features, structures, materials or characteristics may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without conflicting with each other.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.
Claims (10)
- 一种检测类器官最大截面积的方法，其特征在于，所述方法包括：A method for detecting the maximum cross-sectional area of an organoid, characterized in that the method comprises:1)对待测类器官进行可见光断层扫描处理，以便获得多个断层图像；1) performing visible light tomography processing on the organoid to be tested, so as to obtain multiple tomographic images;2)将步骤1)所获得的多个断层图像进行叠加处理，以便获得叠加图像；以及2) superimposing the multiple tomographic images obtained in step 1), so as to obtain superimposed images; and3)基于所述叠加图像，获得待测类器官的最大截面积。3) Obtain the maximum cross-sectional area of the organoid to be tested based on the superimposed image.
- 根据权利要求1所述的方法，其特征在于，所述待测类器官预先培养于基质胶，所述基质胶呈半球状，所述可见光断层扫描是在以下条件的至少之一下进行的：The method according to claim 1, wherein the organoid to be tested is pre-cultured on Matrigel, the Matrigel is hemispherical, and the visible light tomography is carried out under at least one of the following conditions:基质胶的体积与拍摄区间的比值为1μL：150μm；The ratio of the volume of Matrigel to the shooting interval is 1 μL: 150 μm;层间高度为48～52μm或98～102μm；The interlayer height is 48-52 μm or 98-102 μm;在多孔板顶部加入匀光板；Add a dodging plate on top of the perforated plate;LUT值为50～55k。The LUT value is 50-55k.
- 根据权利要求1或2所述的方法，其特征在于，所述可见光断层扫描是用倒置可见光显微镜进行扫描；The method according to claim 1 or 2, characterized in that the visible light tomography is scanned with an inverted visible light microscope;任选地，所述可见光断层扫描是在自然光投射条件下，用倒置金相显微镜进行扫描或钙黄绿素对类器官染色的条件下用倒置荧光显微镜进行扫描；Optionally, the visible light tomography is scanned with an inverted metallographic microscope under the condition of natural light projection or scanned with an inverted fluorescence microscope under the condition of staining the organoid with calcein;任选地，所述倒置荧光显微镜进行断层扫描时激发光为紫外光；Optionally, when the inverted fluorescence microscope performs tomography, the excitation light is ultraviolet light;任选地，所述紫外光的波长为488nm。Optionally, the wavelength of the ultraviolet light is 488nm.
- 一种药物药效评价方法，其特征在于，所述方法包括：A drug efficacy evaluation method, characterized in that the method comprises:对类器官施加待筛选药物；Applying the drug to be screened to the organoid;基于类器官施药后的最大截面积，确认类器官施药后的总最大截面积；Based on the maximum cross-sectional area of the organoid after administration, confirm the total maximum cross-sectional area of the organoid after administration;基于类器官施药后的总最大截面积，确定类器官施药后的总最大截面积的标准化值；以及Determining a normalized value of the total maximum cross-sectional area of the organoid after administration based on the total maximum cross-sectional area of the organoid after administration; and基于所述类器官施药后的总最大截面积的标准化值，确定所述待筛选药物是否为目标药物；Based on the normalized value of the total maximum cross-sectional area of the organoid after administration, determine whether the drug to be screened is the target drug;其中，类器官的最大截面积是通过权利要求1～3任一项所述的方法获得的，总最大截面积是获得的多个类器官最大截面积之和与重复数的比值，所述重复数为预先设置的重复孔的数量；Wherein, the maximum cross-sectional area of the organoid is obtained by the method described in any one of claims 1 to 3, the total maximum cross-sectional area is the ratio of the sum of the maximum cross-sectional areas of multiple obtained organoids to the number of repetitions, and the repeated The number is the number of preset repeated holes;任选地，所述重复孔的数量为3；Optionally, the number of repeated holes is 3;任选地，第n天所述类器官施药后的总最大截面积的标准化值比第0天所述类器官施药后的总最大截面积的标准化值减少，是所述待筛选药物为目标药物的指示，其中，n为不小于1的整数；Optionally, the normalized value of the total maximum cross-sectional area of the organoid after administration on day n is less than the normalized value of the total maximum cross-sectional area of the organoid after administration on day 0, so that the drug to be screened is The indication of the target drug, where n is an integer not less than 1;优选地，第n天所述类器官施药后的总最大截面积的标准化值比第0天所述类器官施药后的总最大截面积的标准化值减少至少30％，是所述待筛选药物为目标药物的指示。Preferably, the normalized value of the total maximum cross-sectional area of the organoid after administration on the nth day is at least 30% less than the normalized value of the total maximum cross-sectional area of the organoid after administration on the 0th day, which is the The drug is an indication of the target drug.
- 根据权利要求1～4任一项所述的方法，其特征在于，所述断层图像是从互不相同的多个截面取得的；The method according to any one of claims 1-4, characterized in that the tomographic image is obtained from a plurality of different sections;任选地，所述断层图像是使用Capture 2.1软件拍摄；Optionally, the tomographic image is taken using Capture 2.1 software;任选地，所述断层图像是使用Capture 2.1软件中的EDF景深扩展功能拍摄。Optionally, the tomographic image is taken using the EDF depth-of-field extension function in Capture 2.1 software.
- 根据权利要求1或4所述的方法，其特征在于，所述叠加图像是基于多张断层图像的吸光值和小波算法进行图像处理获得的；The method according to claim 1 or 4, wherein the superimposed image is obtained by image processing based on the absorbance values of multiple tomographic images and a wavelet algorithm;任选地，所述叠加图像是使用Capture 2.1软件叠加生成。Optionally, the overlay image is overlay generated using Capture 2.1 software.
- 根据权利要求1或4所述的方法，其特征在于，所述类器官最大截面积是使用Image-Pro Plus 6.0和/或Image J软件计算。The method according to claim 1 or 4, wherein the maximum cross-sectional area of the organoid is calculated using Image-Pro Plus 6.0 and/or Image J software.
- 根据权利要求4所述的方法，其特征在于，所述类器官总最大截面积标准化值的变化是使用GraphPad Prism软件统计。The method according to claim 4, wherein the variation of the normalized value of the total maximum cross-sectional area of the organoid is calculated using GraphPad Prism software.
- 根据权利要求4所述的方法，其特征在于，所述类器官总最大截面积的标准化值为类器官第n天的总最大截面积与第0天类器官总最大截面积的比值；The method according to claim 4, wherein the normalized value of the total maximum cross-sectional area of the organoid is the ratio of the total maximum cross-sectional area of the organoid on day n to the total maximum cross-sectional area of the organoid on day 0;任选地，类器官药物药效评价周期为15天；Optionally, the drug efficacy evaluation cycle of organoid drugs is 15 days;任选地，所述药效评价周期内从第0天开始每隔3天对类器官进行所述断层图像扫描。Optionally, the tomographic image scanning of the organoid is performed every 3 days from the 0th day in the drug efficacy evaluation period.
- 根据权利要求1或4所述的方法，其特征在于，所述类器官为肿瘤类器官；The method according to claim 1 or 4, wherein the organoid is a tumor organoid;优选地，所述肿瘤类器官为肠癌类器官。Preferably, the tumor organoid is an intestinal cancer organoid.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101149327A (en) * | 2007-11-06 | 2008-03-26 | 浙江大学 | Antineoplastic drug evaluation and screening method based on cell microscopic image information |
| CN101858866A (en) * | 2010-03-25 | 2010-10-13 | 辽宁大学 | Method for accurately and quantitively detecting effects of anti-prion medicament in living cells |
| US20140365152A1 (en) * | 2013-05-30 | 2014-12-11 | Tech4Imaging Llc | Interactive and Adaptive Data Acquisition System for Use with Electrical Capacitance Volume Tomography |
| CN104403923A (en) * | 2014-11-11 | 2015-03-11 | 南京卡迪奥密生物技术有限公司 | Three dimensional tissue and organ culture model, high throughput automatic stereo image analyzing platform and applications thereof |
| CN105725963A (en) * | 2014-12-07 | 2016-07-06 | 上海市第一人民医院 | Evaluation method for tumour model effective intervention based on optical imaging technology |
| CN106103733A (en) * | 2014-03-20 | 2016-11-09 | 株式会社斯库林集团 | Method of evaluating drug effect and the image processing apparatus for evaluating drug effect |
Family Cites Families (13)
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|---|---|---|---|---|
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| JP3628663B2 (en) * | 2002-02-28 | 2005-03-16 | 独立行政法人科学技術振興機構 | Lightwave coherence tomography system with synthetic light source |
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| WO2008041154A2 (en) * | 2006-10-06 | 2008-04-10 | Koninklijke Philips Electronics N. V. | Spatial characterization of a structure located within an object by identifying 2d representations of the structure within section planes |
| CN103127093B (en) * | 2011-12-02 | 2016-03-02 | 鲁南制药集团股份有限公司 | The purposes of arctigenin in preparation treatment or prevention cerebral glioma medicine |
| WO2014113820A1 (en) * | 2013-01-18 | 2014-07-24 | Shapiro David J | Estrogen receptor inhibitors |
| JP2018174874A (en) * | 2017-04-19 | 2018-11-15 | 国立大学法人 東京医科歯科大学 | Method for measuring water secretion function of epithelial cells |
| JP7160559B2 (en) * | 2017-06-14 | 2022-10-25 | キヤノンメディカルシステムズ株式会社 | MEDICAL IMAGE PROCESSING APPARATUS, MEDICAL IMAGE DIAGNOSTIC APPARATUS, AND MEDICAL IMAGE PROCESSING PROGRAM |
| WO2019046452A1 (en) * | 2017-09-01 | 2019-03-07 | Arizona Board Of Regents On Behalf Of Arizona State University | Thixotropic biocompatible gel for live cell observation in cell computed tomography |
| KR102231835B1 (en) * | 2019-06-13 | 2021-03-25 | 주식회사 휴비츠 | Apparatus and method for tomographic inspection |
| CN110279673B (en) * | 2019-06-21 | 2022-11-25 | 杜彬 | AuNP @ PP/poly (I: C), preparation method thereof and application thereof in preparation of drugs for treating glioma |
| CN210864293U (en) * | 2019-09-06 | 2020-06-26 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Camera chamber for hermetically shooting bacteria and fungus cultures |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101149327A (en) * | 2007-11-06 | 2008-03-26 | 浙江大学 | Antineoplastic drug evaluation and screening method based on cell microscopic image information |
| CN101858866A (en) * | 2010-03-25 | 2010-10-13 | 辽宁大学 | Method for accurately and quantitively detecting effects of anti-prion medicament in living cells |
| US20140365152A1 (en) * | 2013-05-30 | 2014-12-11 | Tech4Imaging Llc | Interactive and Adaptive Data Acquisition System for Use with Electrical Capacitance Volume Tomography |
| CN106103733A (en) * | 2014-03-20 | 2016-11-09 | 株式会社斯库林集团 | Method of evaluating drug effect and the image processing apparatus for evaluating drug effect |
| CN104403923A (en) * | 2014-11-11 | 2015-03-11 | 南京卡迪奥密生物技术有限公司 | Three dimensional tissue and organ culture model, high throughput automatic stereo image analyzing platform and applications thereof |
| CN105725963A (en) * | 2014-12-07 | 2016-07-06 | 上海市第一人民医院 | Evaluation method for tumour model effective intervention based on optical imaging technology |
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| CN113720823A (en) | 2021-11-30 |
| CN113237860A (en) | 2021-08-10 |
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