阅读说明：本技术 检测类器官最大截面积的方法及其应用 (Method for detecting maximum sectional area of organoid and application thereof ) 是由 富国祥 王威 林汉卿 李远闯 李俊强 于 2021-10-22 设计创作，主要内容包括：本发明提出了一种检测类器官最大截面积的方法及其应用,该方法包括：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,该方法能够更方便、准确的检测类器官的最大截面积,从而判断类器官的大小,可进一步评价作为候选药剂的化学物质对类器官的药效作用。(The invention provides a method for detecting the maximum sectional area of an organoid and application thereof, wherein the method comprises the following steps: 1) carrying out visible light tomography processing on the organ to be detected so as to obtain a plurality of tomography images; 2) performing superposition processing on the plurality of tomographic images obtained in the step 1) to obtain a superposed image; and 3) obtaining the maximum sectional area of the organoid to be detected based on the superposed image, wherein the method can more conveniently and accurately detect the maximum sectional area of the organoid so as to judge the size of the organoid and further evaluate the pharmacodynamic action of the chemical substance serving as the candidate medicament on the organoid.)

1. A method of detecting a maximum cross-sectional area of an organoid, said method comprising:

1) carrying out visible light tomography processing on the organ to be detected so as to obtain a plurality of tomography images;

2) performing superposition processing on the plurality of tomographic images obtained in the step 1) to obtain a superposed image; and

3) and obtaining the maximum sectional area of the organoid to be detected based on the superposed image.

2. The method of claim 1, wherein the organoids to be tested are pre-cultured in matrigel, the matrigel having a hemispherical shape, and the visible light tomography is performed under at least one of the following conditions:

the ratio of the matrigel volume to the shooting interval was 1 μ L: 150 μm;

the interlayer height is 48-52 μm or 98-102 μm;

adding a light homogenizing plate at the top of the porous plate;

the LUT value is 50-55 k.

3. The method of claim 1 or 2, wherein the visible light tomography is scanning with an inverted visible light microscope;

optionally, the visible light tomography is scanning with an inverted metallographic microscope under natural light projection conditions or scanning with an inverted fluorescence microscope under conditions of staining organoids with calcein;

optionally, the excitation light of the inverted fluorescence microscope during tomography is ultraviolet light;

optionally, the wavelength of the ultraviolet light is 488 nm.

4. A method for evaluating the efficacy of a drug, the method comprising:

applying a drug to be screened to the organoid;

confirming the total maximum cross-sectional area of the organoids after the organoids are administered based on the maximum cross-sectional area of the organoids after the organoids are administered;

determining a normalized value of the total maximum cross-sectional area after organoid administration based on the total maximum cross-sectional area after organoid administration; and

determining whether the drug to be screened is a target drug based on the standardized value of the total maximum sectional area of the organoids after drug administration;

wherein the maximum cross-sectional area of the organoids is obtained by the method of any one of claims 1 to 3, the total maximum cross-sectional area is a ratio of the sum of the obtained maximum cross-sectional areas of the plurality of organoids to the number of repetitions, which is the number of the previously set repetition holes;

optionally, the number of repeat wells is 3;

optionally, a normalized value of the total maximum cross-sectional area after the organoid administration at day n, which is decreased from the normalized value of the total maximum cross-sectional area after the organoid administration at day 0, is an indication that the drug to be screened is a target drug, wherein n is an integer of not less than 1;

preferably, a normalized value for total maximum cross-sectional area after organoid administration on day n that is at least 30% less than the normalized value for total maximum cross-sectional area after organoid administration on day 0 is indicative of the drug to be screened being the target drug.

5. The method according to any one of claims 1 to 4, wherein the tomographic images are taken from a plurality of cross-sections different from each other;

optionally, the tomograms are taken using Capture 2.1 software;

optionally, the tomogram is taken using an EDF depth extension function in Capture 2.1 software.

6. The method according to claim 1 or 4, wherein the superimposed image is obtained by image processing based on the light absorption values of the plurality of tomographic images and a wavelet algorithm;

optionally, the overlay image is generated using Capture 2.1 software overlay.

7. The method of claim 1 or 4, wherein the organoid maximum cross-sectional area is calculated using Image-Pro Plus 6.0 and/or Image J software.

8. The method of claim 4, wherein the change in the normalized value of total maximum cross-sectional area of the organoid is statistically calculated using GraphPad Prism software.

9. The method of claim 4, wherein the normalized value for total maximum organoid cross-sectional area is the ratio of total maximum organoid cross-sectional area at day n to total maximum organoid cross-sectional area at day 0;

optionally, the organoid drug efficacy evaluation period is 15 days;

optionally, the tomographic scan of the organoids is performed every 3 days from day 0 during the efficacy evaluation period.

10. The method of claim 1 or 4, wherein the organoid is a tumor organoid;

preferably, the tumor organoid is an intestinal cancer organoid.

Technical Field

The invention relates to the field of biomedicine, in particular to a method for detecting the maximum sectional area of an organoid and application thereof.

Background

In recent years, tumors have become the leading cause of death in our population. About 221 million people died of tumors in 2017, accounting for 24.8% of all deaths in the same year. In addition, the tumor belongs to a typical senile disease, and with the aggravation of the aging degree of the population in China, the proportion of the population dying due to the tumor is expected to be greatly increased in the next decades, so that huge pressure and challenge are brought to the society. The tumor treatment mainly comprises means such as surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, endocrine therapy and the like. About 70% of patients need radiotherapy and chemotherapy, but due to individual difference of patients and tumor drug resistance or radiation resistance, the existing clinical tumor drugs have low overall effective rate (about 30%), and unnecessary treatment and ineffective treatment of tumors cause huge waste. Therefore, personalized treatment of tumors is imminent. Accelerate the research and development process of tumor clinical drugs, and screen sensitive treatment population for tumor drugs on the market, which is an important fulcrum for changing the current situation of tumor treatment.

At present, the international main tumor drug research and development models are as follows: tumor cell line model, patient tumor tissue allografting, PDX model. The 2D cell line has low screening cost and high efficiency, but the cell line is an immortalized single type and cannot reflect the heterogeneity and the spatial structure of the tumor and the communication among various cells. Although the human tumor allograft model can truly reflect the in-vivo state of the tumor and retain the heterogeneity of the tumor, the model has the problems of species difference, long manufacturing period, high cost and the like, and many tumors are not successfully established and cannot reflect the medication effect quickly. Since 2014, organoid technology has been the heterophoria. Organoids have incomparable advantages and advances over mainstream drug development technologies.

Organoids (organoids) are multicellular structures that are functionally close to organs and have three-dimensional structures formed by embryonic stem cells, pluripotent stem cells or adult somatic cells in a certain culture environment and under the supporting action of extracellular matrix. Tumor Organoids (PDTOs) refer to a micro-volumetric 3D multicellular structure of primary tumor tissue taken from a tumor Patient in a laboratory culture. Compared with 2D cell line culture, the tumor organoid has a three-dimensional structure, retains the heterogeneity of tumor tissues and is closer to the state of tumors in vivo. Compared with a allograft tumor model, the method has the advantages of high organoid efficiency and low cost, can better realize high-flux drug screening, and can more accurately and rapidly simulate the real drug treatment response condition of a patient.

Since organoids are generally cultured on approximately hemispherical matrigel solids, there can be differences in location and ability to contact growth factors, and organoids themselves have strong heterogeneity, leading to their morphological size and distribution. At present, the method for evaluating the size of organoids is mainly an image analysis method, and the organoids growing on matrigel are photographed by selecting a certain visual field under an optical or fluorescent microscope. And (4) carrying out area and survival rate statistics on each organoid in the output picture so as to judge the size change and cell survival condition of the dosed organoids. Another approach is to grow organoids within specific hydrogel-coated microcavities, growing them as long as possible in one dimension and focal plane, and taking single plane photographs for area and survival statistics, but this approach will greatly reduce organoid heterogeneity. Because of the 3D culture mode, each organoid is located at a different focal plane. Therefore, the inconsistent morphology and limited scalability lead to organoids that are difficult to perform in high-throughput quantification. The existing relative quantitative method only adopts one section shot by an optical or fluorescence microscope for statistics, and cannot accurately indicate the maximum section of each organoid, so that the drug effect of the drug cannot be well indicated. The existing methods can not get rid of the defects under the condition of keeping the original organoid state, and carry out accurate pharmacodynamic analysis.

Disclosure of Invention

The present application is based on the discovery and recognition by the inventors of the following facts and problems:

the inventor finds that the problem that the maximum sectional area of each organoid cannot be accurately indicated by shooting one section can be well solved by performing maximum light intensity projection after the organoids are subjected to continuous tomography scanning by using a microscope and finding out the maximum section of each organoid for quantitative analysis; compared with the traditional single-focal-plane organoid photographic image, the superposed image obtained by continuous tomography is clearer. And (3) calculating the total maximum sectional area of the organoids after overlaying the tomograms, measuring the drug effect result according to the change of the standardized values of the total maximum sectional areas of the organoids of different drug treatment groups, and matching the obtained result with the national standard method result of measuring the drug effect of the organoids. Therefore, the method for calculating the area by continuous tomography of the organoid can accurately evaluate the drug effect of the tumor drug.

To this end, in a first aspect of the invention, the invention proposes a method of detecting the maximum cross-sectional area of an organoid, said method comprising: 1) carrying out visible light tomography processing on the organ to be detected so as to obtain a plurality of tomography images; 2) performing superposition processing on the plurality of tomographic images obtained in the step 1) to obtain a superposed image; and 3) obtaining the maximum sectional area of the organ to be detected based on the superposed image. According to the method for detecting the maximum sectional area of the organoid, the organoid image generated by the method for detecting the maximum sectional area of the organoid is clear, and the number of the organoids observed by shooting only one section is more accurate compared with the conventional method.

According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

according to an embodiment of the present invention, the organoid to be tested is pre-cultured in matrigel, the matrigel is hemispherical, and the visible light tomography is performed under at least one of the following conditions: the ratio of the matrigel volume to the shooting interval was 1 μ L: 150 μm; the interlayer height is 48-52 μm or 98-102 μm; adding a light homogenizing plate at the top of the porous plate; the LUT value is 50-55 k. According to the embodiment of the invention, the smaller the interlayer height setting, the more tomographic images are obtained, the more the superposed images can reflect the real position and size of the organoid, but the smaller the interlayer height setting, the longer the time consumption, and the larger the consumption of a computer and a shooting machine; the problems of uneven illumination, overexposure or too low exposure and the like can cause poor effect of shot images and are not beneficial to superposition of tomographic images, so that the inventor optimizes the shooting interval, the interlayer height, the light path and the exposure intensity of visible light tomographic scanning; according to the embodiment of the invention, the optimized tomography parameters are shot, the obtained organoid images are clear, the light intensity of the tomography images is consistent, the statistical result is more accurate, and the drug effect evaluation result is more real.

According to an embodiment of the invention, the visible light tomography is scanning with an inverted metallographic microscope under natural light projection conditions or scanning with an inverted fluorescence microscope under organoid staining conditions with calcein. According to the embodiment of the invention, only when the organoid is good in shape, the organoid shot by the inverted metallographic microscope can better distinguish the survival and death of the organoid, and a better statistical result is obtained. When the organoids from patients have different forms due to individual differences and the dead or alive state is difficult to judge according to the forms of the organoids shot by an inverted metallographic microscope under a bright field condition, the organoids can be selected to be fluorescently labeled with calcein, the calcein only labels living cells, and the fluorescence microscope is used for detecting the maximum sectional area of the organoids. According to the embodiment of the invention, when the organoid has a good shape, the visible light tomography has no limitation on the visible light microscope, and any visible light microscope capable of accurately observing can be used.

According to the embodiment of the invention, the exciting light is ultraviolet light when the inverted fluorescence microscope carries out tomography.

According to an embodiment of the invention, the wavelength of the ultraviolet light is 488 nm.

In a second aspect of the present invention, there is provided a method for evaluating drug efficacy, the method comprising: applying a drug to be screened to the organoid; confirming the total maximum cross-sectional area of the organoids after the organoids are administered based on the maximum cross-sectional area of the organoids after the organoids are administered; determining a normalized value of the total maximum cross-sectional area after organoid administration based on the total maximum cross-sectional area after organoid administration; and determining whether the drug to be screened is a target drug based on the normalized value of the total maximum cross-sectional area after the organoid is administered; wherein the maximum cross-sectional area of the organoids is obtained by the method according to the first aspect, the total maximum cross-sectional area is a ratio of the sum of the obtained maximum cross-sectional areas of the plurality of organoids to the number of repetitions, which is the number of the preset repetition holes. According to the embodiment of the invention, the organoid image generated by the method for detecting the maximum sectional area of the organoid is clear, the number of observable organoids is more accurate than that of the organoid image generated by shooting one section, and the obtained evaluation result of the drug effect of the tumor drug has high consistency with the clinical reaction of a patient after the drug is taken.

According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

according to an embodiment of the invention, the number of said repeating holes is 3. According to the embodiment of the invention, the organoid to be tested is cultured in matrigel in advance, the matrigel is placed in culture holes of a 48-hole plate, when m culture holes are subjected to the same treatment, the m culture holes are the same treatment repeated holes, the number of the treatment repeated holes is m, wherein m is an integer larger than 0.

According to an embodiment of the present invention, a decrease in the normalized value of the total maximum cross-sectional area after the organoid is administered at day n from the normalized value of the total maximum cross-sectional area after the organoid is administered at day 0 is an indication that the drug to be screened is a target drug, wherein n is an integer of not less than 1. According to an embodiment of the invention, the day of organoid administration is day 0; normalized value is the ratio of the total maximum cross-sectional area of the organoid after administration on day n to the total maximum cross-sectional area of the organoid after administration on day 0, e.g., the normalized value for total maximum cross-sectional area of the organoid on day 1 is: s1 is total maximum cross-sectional area after organoid administration on day 1/total maximum cross-sectional area after organoid administration on day 0, S2 is total maximum cross-sectional area after organoid administration on day 2/total maximum cross-sectional area after organoid administration on day 0, and S3 is total maximum cross-sectional area after organoid administration on day 3/total maximum cross-sectional area after organoid administration on day 0.

According to an embodiment of the invention, a normalized value of the total maximum cross-sectional area after the organoid administration on day n that is at least 30% less than the normalized value of the total maximum cross-sectional area after the organoid administration on day 0 is indicative of the drug to be screened being a target drug. According to the embodiment of the invention, the standardized value of the maximum sectional area of the organoid is monitored for 15 days for the drug to be detected, wherein the standardized value of the organoid area is detected once every 3 days, if cancer cells are killed and the activity is reduced due to the pharmacodynamic action of the drug, the results of reducing the area of the tumor organoid and the standardized value of the maximum sectional area of the tumor organoid can be obtained, the standardized value of the total maximum sectional area of the organoid is calculated after the maximum sectional area of the organoid is obtained through verification, and the obtained drug efficacy result has high consistency with the clinical reaction of a patient after the drug is taken by the patient according to the change of the standardized value of the total maximum sectional area of the organoid.

According to an embodiment of the present invention, the tomographic images are taken from a plurality of cross-sections different from each other. According to the embodiment of the invention, visible light progressive tomography scanning is carried out after the top and bottom sections of the organoid hemisphere matrigel are set, wherein the parameters of the photographed sections and the interlayer heights can be set according to experimental requirements, and when the photographed sections of the tomography images are selected, the sections can be used for photographing organoids as much as possible under the condition of clear images, so that the positions and the sizes of the organoids are reduced to the maximum extent.

According to an embodiment of the present invention, the tomograms are taken using Capture 2.1 software.

According to the embodiment of the invention, the tomogram is shot by using an EDF depth expansion function in the Capture 2.1 software. According to the embodiment of the invention, the method has no limitation on shooting software, and any software capable of accurately carrying out tomography can be used.

According to the embodiment of the invention, the superimposed image is obtained by image processing based on the light absorption values of the plurality of tomographic images and a wavelet algorithm. According to the embodiment of the invention, the method has no limitation on software for obtaining the superposed image, and any software capable of performing image processing based on the light absorption value of the tomographic image and the wavelet algorithm can be used.

According to an embodiment of the invention, the overlay image is generated using a Capture 2.1 software overlay.

According to an embodiment of the invention, the maximum organoid cross-sectional area is calculated using Image-Pro Plus 6.0 and/or Image J software. According to the embodiment of the invention, the method has no limitation on the software for calculating the maximum sectional area of the organoid, and any software capable of calculating the maximum sectional area of the organoid in the image can be used.

According to an embodiment of the invention, the variation of the normalized value of the total maximum cross-sectional area of the organoid is counted using GraphPad Prism software. According to the embodiment of the invention, the statistics by using the GraphPad Prism software refers to the drawing demonstration by using the GraphPad Prism software, the method is not limited by software for drawing the change of the standardized value of the total maximum sectional area of the organoid, and any software capable of drawing the change of the standardized value of the total maximum sectional area of the organoid can be used.

According to an embodiment of the invention, the normalized value for the total maximum cross-sectional area of the organoid is the ratio of the total maximum cross-sectional area of the organoid at day n to the total maximum cross-sectional area of the organoid at day 0.

According to the present example, the organoid drug efficacy evaluation period was 15 days.

According to an embodiment of the present invention, the tomographic imaging of the organoids is performed every 3 days from day 0 in the efficacy evaluation period. According to the embodiment of the invention, the organ area standard value of the drug to be detected is monitored for 15 days, wherein the organ area standard value is detected once every 3 days, and the obtained drug effect result of the intestinal cancer drug has high consistency with the clinical response of a patient after the drug is taken.

According to an embodiment of the invention, the organoid is a tumor organoid.

According to an embodiment of the invention, the tumor organoid is an intestinal cancer organoid.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a flow chart of a colon cancer organoid culture according to an embodiment of the present invention; digesting the tumor tissue of the operation or biopsy of a tumor patient into a cell suspension, culturing the cell suspension in matrigel, and growing into a tumor organoid with a three-dimensional structure;

FIG. 2 is a design drawing of an experiment of a drug experiment of an intestinal cancer drug by an intestinal cancer organoid according to an embodiment of the present invention; planting organoids in a 48-pore plate, respectively adding 5-fluorouracil and irinotecan for treatment when the diameter reaches 100 mu m, and replacing the culture medium every three days for 15 days;

FIG. 3 is a flow chart of slice photographing and an exemplary diagram of a generated overlay image during a process of a drug experiment of an intestinal cancer drug by an intestinal cancer organoid according to an embodiment of the present invention;

FIG. 4 is a graph showing the trend of the total maximum sectional area of the intestinal cancer organoids in the Z-axis of the change in the inter-layer height of the intestinal cancer organoids in the inverted metallographic microscope;

FIG. 5 is a graph showing the comparison of the statistical circling results of the cross-sectional areas of 50 μm (left) and 150 μm (right) between the Z-axis images of the colon cancer organs according to the embodiment of the present invention by using the inverted metallographic microscope; using ImageJ for selection, 150 μm lost signals for many small organoids compared to 50 μm;

FIG. 6 is a sectional image contrast of an organ of bowel cancer with an inverted metallographic microscope for continuous tomographic scanning with modified optical paths viewed from the front (left) and back (right) of the organ of bowel cancer according to an embodiment of the invention;

FIG. 7 is a contrast image of sectional images of a colon cancer organoid observed under high (left) and low (right) exposure conditions using continuous tomographic scanning with an inverted metallographic microscope according to an embodiment of the present invention;

FIG. 8 is a graph showing the trend of the total maximum cross-sectional area with Z-axis photographing of the change in the inter-layer height in the case of continuous tomographic scanning using a fluorescence microscope for an intestinal cancer organoid according to an embodiment of the present invention;

FIG. 9 is a photograph of single focal plane organoids taken by inverted metallographic microscope single layer scan at day 0 (left) and day 6 (right) from an intestinal cancer organoid according to an embodiment of the invention;

FIG. 10 is a superposition of multiple Z-Stack focal planes at day 0 (left) and day 6 (right) obtained by sequential tomography of intestinal cancer organoids using an inverted metalloscope according to an embodiment of the present invention;

FIG. 11 is a graph showing the trend of the total maximum cross-sectional area of the colon organs after treatment with 5-fluorouracil and irinotecan, obtained by area method using an inverted metallographic microscope with continuous tomography of the colon organs according to an embodiment of the present invention;

fig. 12 is a cross-sectional overlay image of fluorescence marker obtained by continuous cross-sectional scanning of intestinal cancer organoids with a fluorescence microscope by an area method according to an embodiment of the present invention, in which single organoids having different sizes and shapes are enclosed by a dotted frame;

FIG. 13 is a plot of the trend of total maximum cross-sectional area of colon organs after treatment with 5-fluorouracil and irinotecan, obtained by area method using fluorescence microscopy on serial sections of colon cancer organoids in accordance with an embodiment of the present invention;

FIG. 14 is a fluorescence-labeled tomographic image obtained by continuous tomographic scanning of an intestinal cancer organoid with a high power microscope according to a three-dimensional modeling method of an embodiment of the present invention; 56 continuous images are obtained in the shooting interval, the change of the three-dimensional shape of the organoid can be clearly observed, from the initial non-fluorescence signal to the gradual shooting of the upper half part, the central part and the lower half part of the organoid until the signal is lost;

FIG. 15 is an exemplary diagram of three-dimensional modeling of fluorescence-labeled tomographic images obtained by sequential tomographic scanning of intestinal cancer organs using a high power microscope using MATLAB according to the three-dimensional modeling method of the present invention; the upper left is a cross section diagram in the X-axis direction, the lower left is a vertical plane diagram in which the maximum section in the Z-axis direction is located, the upper right is a longitudinal section diagram in the Y-axis direction, the lower right is a vertical plane organoid sorting result in which the maximum section in the Z-axis direction is located, and each organoid with different shapes and sizes is circled by a broken line frame;

FIG. 16 is a graph of total surface area of colon cancer organoids after treatment with 5-fluorouracil and irinotecan, obtained by continuous tomography of colon cancer organoids using high power microscope, according to the three-dimensional modeling method of the present invention; and

FIG. 17 is a graph comparing the results of evaluating the efficacy of 5-fluorouracil and irinotecan according to the present invention in colon cancer organoid area method with the results of response after clinical application to patients, wherein the organoid efficacy results are classified as sensitive and insensitive (note: some patients have organoids not treated with irinotecan according to their clinical application, i.e., patients have not clinically used irinotecan); the results of clinical patients are divided into two categories of good response and poor response; as can be seen in the figure, most of the patients with good clinical response have sensitive organoid drug effect and poor response, the organoid drug effect result is mostly insensitive, and the drug effect measured by an area method has high consistency compared with the clinical result, wherein the sensitivity is 81.03%, the specificity is 92.31%, and the accuracy is 86.36%.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

In the following examples, the intestinal cancer organoids are used as a model, and the size change of the organoids in the matrigel in the whole drug administration period process after the tumor drug is added is accurately judged by using an advanced photomicrography technology, so that the drug effect of the tumor drug is judged, and clinical drug administration is guided.

In some embodiments, the inventors scan with an inverted metallographic microscope or an inverted fluorescence microscope under conditions in which calcein stains organoids. Only when the organoid is good in shape, the organoid shot by the inverted metallographic microscope can better distinguish the survival and death of the organoid, and a better statistical result is obtained. When the organoids from patients have different forms due to individual differences and the dead and alive states of the organoids are difficult to judge by taking the organoids through an inverted metallographic microscope, the organoids can be selected to be fluorescently labeled with calcein, the calcein only labels living cells, and the maximum cross-sectional area of the organoids is detected by using the fluorescence microscope. When the inverted fluorescence microscope is used for carrying out tomography, exciting light is ultraviolet light; the wavelength of the ultraviolet light used was 488 nm.

In some embodiments, there are 3 replicate wells per treatment group, each culture well having multiple organoids, and for well-formed organoids that can be imaged visible light using an inverted metallographic microscope, tomographic scanning can be performed directly using the inverted metallographic microscope; when the organoids from patients have different forms due to individual differences and the dead and alive states of the organoids are difficult to judge by taking the organoids through an inverted metallographic microscope, the organoids can be selected to be fluorescently labeled with calcein, the calcein only labels living cells, and the maximum cross-sectional area of the organoids is detected by using the fluorescence microscope.

In some embodiments, the tomographic image in each culture well is used to generate a superimposed image, the position and maximum cross-sectional area of each organoid in the matrigel can be accurately found out by using the superimposed image, the sum of the maximum cross-sectional areas of all organoids in a single culture well is obtained, and then the 3 maximum cross-sectional areas obtained from 3 repeated wells of the same treatment group are added again and averaged, which is referred to as the total maximum cross-sectional area of organoids in the present invention. The total maximum sectional area of the organoid is calculated according to the maximum sectional area of the organoid, and the standardized value of the total maximum sectional area of the organoid is calculated according to the total maximum sectional area of the organoid, so that the size of the organoid is evaluated, and the size of the organoid can reflect the drug effect of the drug. The method eliminates statistical errors caused by the position, distribution, size, form and heterogeneity differences of organoids in matrigel; in the whole dosing period, under the condition of keeping the original state of the organoid, different time nodes can be selected for statistics, and the size difference of the organoid at different time points is observed, so that the drug effect after dosing is accurately judged, and a basis is provided for clinical diagnosis and medication. The clinical drug effect consistency verification proves that the evaluation result of the drug effect measured by the method is consistent with the clinical drug effect evaluation result, so the method is the most accurate method for judging the drug effect of the tumor organoid drug at present.

The embodiments will be described in detail below.

EXAMPLE 1 intestinal cancer organoid culture

The collected fresh biopsy tumor samples of intestinal cancer were photographed and specific information was recorded, and after comparison, they were washed 5 times with pre-cooled PBS (100 Xpenicillin/streptomycin was added), 5min each time. On a sterile bench, the sterile petri dish was placed on ice and an appropriate amount of pre-chilled sterile PBS was poured into it, and the cancer tissue was placed in it, and the tumor sample was cut into small pieces with a sterile razor blade and transferred into 8mL of a digested solution preheated at 37 ℃. The formula of the digestive juice is as follows: 7mL of DMEM medium (GIBCO, C1199500BT), 500U/mL of collagenase IV (Sigma-aldrich, C9407), 1.5mg/mL of collagenase II (Solardio, C8150), 20. mu.g/mL of hyaluronidase (Solardio, h8030), 0.1mg/mL of neutral protease II (Sigma-aldrich, D4693), 10. mu.M of RHOK inhibitor ly27632(Sigma-aldrich, Y0503) and 1% fetal bovine serum. Digesting with shaking table at 37 deg.C for 40 min. After digestion was completed, 200g was centrifuged for 5min and washed 5 times with pre-cooled sterile PBS, 5min each. All cell pellets were then pelleted by centrifugation at 200g (20. mu.L under a lens before centrifugation and cell numbers were counted). After extraction, a proper amount of matrigel (corning, #356231) was added to a precooled 1mL tip to make the number of cell clusters in each 50. mu.L of matrigel about 200, and then the mixture was suspended and mixed uniformly by a 200. mu.L precooled tip and inoculated into a preheated 24-well plate with 50. mu.L of each well. And placing the inoculated culture plate in a constant-temperature incubator at 37 ℃ for incubation for 5-8min, and taking out. mu.L of organoid medium (Danwang medical, Cat # K20001) was added to each well, and the inoculation was observed under a microscope, followed by culturing in an incubator. The organoid growth was observed daily and recorded by taking pictures with a microscope, and the medium was changed every 3 days, the overall procedure being shown in FIG. 1.

Passage was once for 1-2 weeks. And (3) absorbing the culture medium in the culture hole during passage, adding 500mL of precooled PBS, placing the culture plate on an ice box for a moment, blowing off the matrigel by a precooling gun head, moving the culture plate into a 15mL sterile centrifuge tube, blowing and beating the culture plate to completely blow off and uniformly mix the matrigel, centrifuging the culture plate for 5min by a centrifuge with the temperature of 4 ℃ of 69g, and absorbing the supernatant and the matrigel precipitated at the bottom. And adding precooled PBS to repeatedly wash for three times to remove the matrigel. And adding 5mL of precooled PBS to resuspend the tube bottom organoids, blowing and beating the organoids into cell clusters (observing that the cell clusters are blown and beaten into single cells or cell clusters under a mirror) for 50-100 times, and centrifuging for 5min at the temperature of 4 ℃ by a centrifuge of 69 g. The culture was continued according to organoid culture protocol, and the cells were inoculated at a ratio of 1 to 4 per well at the time of subculture, with the medium composition shown in Table 1.

Table 1: intestinal cancer organoid medium component table

Example 2 inverted metallographic microscope imaging organoid parameter optimization

Good cultured organoids were selected, digested and passaged to 48-well plates as in example 1. The initial graft organoid density was 10-15/. mu.L matrigel, and 300. mu.L of medium was added per well. Finally, 200 +/-50 organoids are contained in 10 mu L of matrigel in each hole, a parameter optimization experiment is carried out when the diameters of the organoids reach 100 mu m, 3 repeated holes are arranged in total, the shooting period is 15 days, the organoids in each hole are optically imaged every 3 days from the 0 th day, and progressive tomography is carried out by combining the EDF depth of field extension function of Capture 2.1 shooting software with an inverted metallographic microscope to output a plurality of Z-Stack images. The shooting software superposes a plurality of Z-Stack images by using a wavelet algorithm according to the light absorption value of each layer to generate a complete superposed image containing all the shot organoids, and the specific operation flow is shown in FIG. 3.

1. Shooting interval optimization

Since matrigel is a hemispherical solid unlike cells, it is necessary to determine the top and bottom regions of the matrigel shot, i.e., where to start and where to end the shot. Through the optimization of the parameters, the inventor obtains that the shooting interval of 10 μ L of the hemispherical matrigel is about 1500 μm.

2. Shot layer height optimization

The height between layers, namely when in tomography, the final output image is optimized by selecting how many microns of pictures are taken in the shooting interval. The inventors set the heights of the imaging layers of different Z-axes to 10, 25, 50, 75, 100 and 150 μm, and the specific results are shown in fig. 4 and 5. As can be seen from FIG. 4, the total cross-sectional area of the obtained organoid becomes smaller and smaller as the interlayer height of the Z-axis photographing increases, and the area counting circled shown in FIG. 5 shows that many small organoid signals are lost when the interlayer height is 150 μm compared with 50 μm; 10. the statistical results of the areas of 25 and 50 mu m are basically not different; further, the smaller the interlayer height setting, the longer the time consumption, and the larger the computer and photographing equipment wear. Therefore, combining the above results, the inventors selected 50 μm as the set value of the interlayer height.

3. Light path and exposure

When a high content instrument on the market at present shoots a perforated plate (flat bottom plate) for culturing organoids or cells, a condenser lens emits parallel light alpha, light refraction is formed inside the perforated plate, and a beta included angle is generated, so that the phenomenon of uneven illumination is caused; the organoids near the edge of the well plate cannot be clearly observed. Thus, the inventors changed the light path and added a light homogenizing plate on top of the 48-well plate used to disturb the parallel light path into uniformly scattered light, as shown in FIG. 6. In order to output an accurate statistical image, the optimal exposure level needs to be determined so as to unify the light intensity used in the superposition, and overexposure or too low exposure can cause poor effect of the shot image and interfere with the statistical result. After screening, the inventors found that setting the lut (look up table) in the range of 50-55k works best, and the resulting image is shown in fig. 7.

Example 3 fluorescence microscopy imaging organoid interlayer height parameter optimization

Good cultured organoids were selected, digested and passaged to 48-well plates as in example 1. The initial graft organoid density was 10-15/. mu.L matrigel, and 300. mu.L of medium was added per well. Finally, 200 +/-50 organoids are contained in 10 mu L of matrigel in each hole, a parameter optimization experiment is carried out when the diameters of the organoids reach 100 mu m, 3 repeated holes are arranged in total, the shooting period is 15 days, the organoids in each hole are optically imaged every 3 days from the 0 th day, and progressive tomography is carried out by combining the EDF depth of field extension function of Capture 2.1 shooting software with an inverted fluorescence microscope to output a plurality of Z-Stack images. The shooting software superposes a plurality of Z-Stack images by using a wavelet algorithm according to the light absorption value of each layer to generate a complete superposed image containing all the shot organoids, and the specific operation flow is shown in FIG. 4.

The inventors fluorescently labeled organoids with calcein, which only labeled living cells. Calcein labeling and fluorescence imaging were performed on multiple organoids per well every 3 days starting on day 0 during 15 of the culture cycle. Calcein with a final concentration of 4 μ M is added into the culture medium for staining for 60min, and an EDF depth of field extension function of Capture 2.1 shooting software is combined with an inverted fluorescence microscope to display a green fluorescence picture by 488nm excitation light. The inventors screened the Z-axis shot interlayer heights and set the interlayer heights to 25, 50, 100, 150, and 200 μm, and the results showed that the shot layer height of 10 μ L of the hemispherical matrigel was preferably set to 100 μm, and the remaining parameters were the same as those of example 2, and the specific results are shown in fig. 8.

Example 4 continuous tomography vs. conventional single slice images

The organoids obtained in example 2 were used to obtain conventional single-field images using an inverted metallographic microscope, and superimposed images obtained by tomographic scanning according to the parameters in example 2. The picture obtained by single-layer shooting often contains a plurality of virtual focal plane organoids, so that the survival condition of the organoids cannot be observed, the area statistics is difficult, and the error of the experimental result is large. In the 0 th day, finding a visual field with most of the organoids in the same shooting focal plane, and shooting by using a single focal plane; on day 6, in the same visual field as day 0, the organoids are cultured and enlarged, the focal plane becomes non-uniform, as shown in fig. 9, the organoids which cannot be focused form a light spot ghost during imaging, so that the number of observable organoids is lost, and an error is formed. The defects are well avoided in continuous tomography, the generated image organoid is clear, and area statistics is convenient. On day 0, finding a visual field with most of the organoids in the same shooting focal plane, shooting by using a Z-stack multilayer focal plane, and synthesizing into a superposed image by using an EDF function. On day 6, the same procedure was used to capture images, which better enable multiple organoids at different focal planes to be observed in a single overlay image, as shown in FIG. 10.

Example 5 verification of evaluation of drug efficacy by area method

Good cultured organoids were selected, digested and passaged to 48-well plates as in example 1. The initial graft organoid density was 10-15/. mu.L matrigel, and 300. mu.L of medium was added per well. Finally, 10. mu.L of matrigel per well contained 200. + -.50 organoids, which were treated with drugs when the organoids reached 100. mu.m in diameter. 2 experimental groups including 5-fluorouracil (5-Fu) group and irinotecan (CPT-11) group. 10mM 5-Fu (Selleck, S1209) or 10mM CPT-11(Selleck, S2217) was added to the culture medium of the organoids of the experimental group, respectively, and after 3 days the medium was replaced with the drug-added medium, followed by replacement every 3 days with the drug-free medium. The procedure of 15-day culture was followed from the time of drug addition, as shown in FIG. 2, and the following experimental procedures were carried out on the obtained organoids after 15-day culture as described in example 2:

1. evaluation of drug efficacy by area method

1) Inverted metallographic microscope for imaging organoids

This experiment was set up with 2 experimental groups, each treatment group was set up with 3 replicate wells.

In 15 days after the addition of the drugs, the organoids of each well treated with the two drugs 5-Fu and CPT-11 were optically imaged every 3 days from day 0, and the specific operation procedures and parameter settings were the same as in example 2.

The total maximum cross-sectional area of each treatment group was obtained by measuring the number of organoids and the maximum cross-sectional area of each organoid in the overlay Image of each culture well using Image-Pro Plus 6.0(Media Cybernetics, Inc.) or Image J software (National Institute of Health, USA), adding the maximum cross-sectional areas of multiple organoids in each culture well, and calculating the average of the sums of the maximum cross-sectional areas of 3 replicate culture wells in each treatment group. The total maximum sectional area of the organoids on day 0 was normalized to 100%, and the ratio of the total maximum sectional area of the organoids counted on day 1 and the following days to the total maximum sectional area of day 0 was the normalized value, i.e., the percentage of the total maximum sectional area of the organoids every 3 days in the culture period was obtained, and the specific results are shown in table 2. The total maximum cross-sectional area change before and after dosing of the organoids under different treatment modalities was made with GraphPad Prism, as shown in fig. 11. The drug effect of the tumor drug is deduced according to the change result of the standardized value of the total maximum sectional area of the organoids after the drug is added, and the tumor drug is used for clinical medication guidance.

Table 2:

number of days	5-fluorouracil group	Irinotecan group
			0	100±8.2	100±7.5
3	110±10.7	147±8.9
			6	80±7.2	50±11.7
9	60±8.1	40±6.6
			12	48±5.0	20±4.8
15	36±6.2	16±8.5

2) Inverted fluorescence microscope for organoid imaging

The experiment was set up in 2 experimental groups with 18 wells per treatment group.

The inventors fluorescently labeled organoids with calcein, which only labeled living cells. Calcein labeling and fluorescence imaging were performed on each of the 3 well organoids treated every 3 days starting from day 0, 15 days after dosing. Calcein with a final concentration of 4 μ M is added into the culture medium of 3 wells for each detection to stain for 60min, and the stained organoids are not used; the depth of field extension function of EDF using Capture 2.1 shooting software was combined with an inverted fluorescence microscope, green fluorescence was visualized with 488nm excitation light, the specific operation flow and parameter settings were consistent with those of example 3, and the obtained fluorescence-labeled tomographic overlay image was shown in fig. 13.

The total maximum cross-sectional area of each treatment group was obtained by measuring the number of organoids and the maximum cross-sectional area of each organoid in the overlay Image of each culture well using Image-Pro Plus 6.0(Media Cybernetics, Inc.) or Image J software (National Institute of Health, USA), adding the maximum cross-sectional areas of multiple organoids in each culture well, and calculating the average of the sums of the maximum cross-sectional areas of 3 replicate culture wells in each treatment group. The total maximum sectional area of the organoids on day 0 was normalized to 100%, and the ratio of the total maximum sectional area of the organoids counted on day 1 and the following days to the total maximum sectional area of day 0 was the normalized value, i.e., the percentage of the total maximum sectional area of the organoids every 3 days in the culture period was obtained, and the specific results are shown in table 3. The total maximum cross-sectional area change before and after dosing of the organoids under different treatment modalities was made with GraphPad Prism, as shown in fig. 13.

Table 3:

number of days	5-fluorouracil group	Irinotecan group
			0	100±7.5	100±6.5
3	123±11.7	167±6.8
			6	88±8.2	64±10.4
9	66±8.1	44±8.6
			12	51±6.4	31±5.7
15	37±5.2	24±8.5

2. Three-dimensional modeling method for evaluating drug efficacy

The experiment was set up in 2 experimental groups with 18 wells per treatment group.

The inventors fluorescently labeled organoids with calcein, which only labeled living cells. Calcein labeling and fluorescence imaging were performed on each of the 3 well organoids treated every 3 days starting from day 0, 15 days after dosing. Calcein with a final concentration of 4 μ M is added into the culture medium of 3 wells for each detection to stain for 60min, and the stained organoids are not used; the organoids were tomographically imaged using a high power microscope with fluorescence stained organoids combined with 40 times or more of the high power microscope capable of filtering stray light, as shown in fig. 14.

The obtained tomographic image was three-dimensionally modeled using MATLAB, and the surface area of the organ after the drug addition was counted using the three-dimensional modeling as shown in fig. 15. The total organoid surface area on day 0 was normalized to 100% and the ratio of the total surface area counted on the following days to the total surface area on day 0 was normalized to obtain the percentage of total organoid surface area every 3 days in the culture cycle, as shown in table 4. Graph of the variation results of the normalized values of the total area before and after dosing of the organoids in different treatment modes was prepared using GraphPad Prism, as shown in fig. 16. The drug effect of the tumor drug is deduced according to the result of the standardized change of the total surface area of the organoids with time after the drug is added, and the tumor drug is used for clinical medication guidance.

The three experimental results show that the maximum section change of the organoid is counted by overlaying the organoid through a fluorescence microscope after the inverted metallographic microscope is utilized by adopting an area method or the fluorescence labeling so as to evaluate the drug effect, the surface area change of the organoid is counted by adopting a three-dimensional modeling method so as to evaluate the drug effect, and the drug effect evaluation results obtained by the two methods are consistent.

Table 4:

number of days	5-fluorouracil group	Irinotecan group
			0	100±10.2	100±9.4
3	177±12.7	201±10.7
			6	145±8.3	84±11.9
9	77±8.0	56±7.5
			12	62±7.1	35±5.8
15	36±6.0	21±8.0

Example 6 verification of consistency between efficacy measured by area method and clinical efficacy

The inventor adopts an area method to measure the sensitivity of organoids derived from 110 intestinal cancer patients to clinical drugs of 5-fluorouracil and irinotecan, and carries out consistency verification with the response of the patients after clinical medication. As shown in FIG. 17, organoid drug efficacy results were classified into sensitive and non-sensitive (note: some patients 'organoids were not treated with irinotecan according to their clinical drug use, i.e., clinical patients did not use irinotecan either), and clinical patients' drug efficacy results were classified into two categories, good response and poor response; as can be seen from the figure, the patients with good clinical response mostly have sensitive organoid drug effect, and the patients with poor response mostly have insensitive organoid drug effect. Compared with clinical results, the medicine effect measured by an area method has high consistency, the sensitivity is 81.03 percent, the specificity is 92.31 percent, and the accuracy is 86.36 percent. This indicates that in subsequent clinical trials and clinical practice, performing organoid susceptibility testing involving more drugs not only can find truly effective therapeutic drugs for patients, but also suggests patients who have not found effective drugs to try new treatment modalities or strategies.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.