阅读说明：本技术 一种水凝胶包被的微组织块培养药敏检测方法 (Hydrogel-coated microtissue block culture drug sensitivity detection method ) 是由 何远桥 陈传军 于 2018-06-21 设计创作，主要内容包括：本发明公开了一种水凝胶包被的微组织块培养药敏检测方法,还可以称为“果冻”药敏检测法,其主要包括以下步骤：肿瘤标本采集,肿瘤样本处理,组织块接种,药物处理,敏感性检测和和结果分析,分析时根据吸光度值换算公式进行换算得出,将孔板各孔的吸光度值进行换算,进而计算出每种药物对肿瘤组织块活性的影响。本发明保存了肿瘤的异质性和微环境,操作简单,实验条件要求低,可以快速、准确的筛选出最适合患者的肿瘤化疗方案,本发明的方法也可以也可以用于移植瘤小鼠(含PDX模型)肿瘤组织的微组织块培养和药敏检测,还可用于检测正常组织对各种干预措施的反应,如正常肝脏组织毒理学检测。(The invention discloses a hydrogel-coated microtissue block culture drug sensitivity detection method, which can also be called a 'jelly' drug sensitivity detection method and mainly comprises the following steps: collecting a tumor specimen, processing a tumor sample, inoculating a tissue block, processing a medicament, detecting sensitivity and analyzing results, converting according to an absorbance value conversion formula during analysis, converting the absorbance value of each hole of the pore plate, and further calculating the influence of each medicament on the activity of the tumor tissue block. The method has the advantages of tumor heterogeneity and microenvironment preservation, simple operation and low experimental condition requirement, can quickly and accurately screen out the tumor chemotherapy scheme most suitable for patients, can also be used for micro tissue block culture and drug sensitivity detection of tumor tissues of transplanted tumor mice (containing PDX models), and can also be used for detecting the response of normal tissues to various intervention measures, such as normal liver tissue toxicology detection.)

1. A hydrogel-coated microtissue block culture drug sensitivity detection method is characterized by comprising the following steps:

a) collecting a tumor specimen: cutting tumor tissue with vigorous growth and no ulcer necrosis at the edge of the tumor during operation, rapidly placing the tumor tissue into a centrifuge tube containing tissue preservation solution, and placing the centrifuge tube into an ice bag for low-temperature transportation;

b) tumor sample treatment: detecting tumor tissue activity with 0.4% trypan blue dye solution in laboratory biosafety cabinet, removing tissue without activity, soaking tissue in 0.2% iodophor solution for 30 s for sterilization, washing tissue with 3% double-antibody-containing PBS, and dividing tumor tissue into 2 × 2 × 2mm³Placing a cut tissue block in each hole of a 96-hole cell culture plate, and placing the cell culture plate on ice or a blue ice brick for precooling;

c) tissue block inoculation: adding a certain amount of 4 ℃ precooled I type collagen solution into a cell culture hole containing a tissue block, submerging all the tissue block, then covering the cell culture hole, putting the cell culture hole into a cell culture box with the carbon dioxide content of 5% at 37 ℃ for incubation for 2-3 hours, taking out a culture plate after collagen is solidified, adding a proper amount of tissue culture medium into a biological safety cabinet, and then putting the culture plate into the cell culture box for culture;

d) and (3) drug treatment: after the tissue block is cultured in a cell culture box for 24 hours, the original culture medium is removed, a complete culture medium containing the test drug is added, and the complete culture medium containing the test drug is replaced every 24 hours;

e) and (3) sensitivity detection: after the tissue block is cultured for 3 to 7 days by using complete culture medium medicine containing a test medicine, absorbing and removing the medicine-containing culture medium, washing the tissue block for 3 times by using PBS buffer solution, then adding MTT solution, absorbing the MTT solution after 6 hours, taking the tissue block out of hydrogel by using an ophthalmic forceps, putting the tissue block into a new cell culture hole, adding 200ul DMSO reagent, shaking for 10min, absorbing 100ul solution from each hole plate in a cell culture plate filled with the tissue block, adding the absorbed solution into the hole plate of the new cell culture plate, and detecting the absorbance value of each hole by using a microplate reader;

f) and (4) analyzing results: and (4) converting the absorbance value of each hole of the pore plate according to an absorbance value conversion formula, and further calculating the influence of each drug on the activity of the tumor tissue mass.

2. The hydrogel-coated microtissue block culture susceptibility testing method of claim 1 further comprising: in step a), the collected tumor tissue is cut from a part with relatively vigorous tumor growth by a doctor during tumor resection operation, the resected tumor tissue is put into a 50ml centrifuge tube containing a tissue preservation solution, the ice bag is transported to a laboratory at low temperature, and the time from the separation of the specimen to the low-temperature transportation to the laboratory is not more than 48 hours at most, preferably within 2 hours.

3. The hydrogel-coated microtissue block culture susceptibility testing method of claim 1 further comprising: in the step c), the adopted cell culture plate is a 96-hole, 48-hole or 24-hole plate, and the added type I collagen solution can be replaced by Matrigel gel or other temperature-sensitive hydrogel; during incubation, the pore plate is placed in a cell culture box, the temperature is controlled at 37 ℃, incubation is carried out for 2-3 hours, a proper amount of tissue culture medium is added into each pore after hydrogel is solidified, and then the culture plate is continuously placed in the cell culture box for culture.

4. The hydrogel-coated microtissue block culture susceptibility testing method of claim 1 further comprising: in step e), the MTT solution used may be replaced with a detection reagent such as CCK reagent, MTS reagent, or the like.

Technical Field

The invention relates to the technical field of tumor drug sensitivity detection, in particular to a hydrogel-coated microtissue block culture drug sensitivity detection method.

Background

In 2018, 2 months, national cancer centers released the latest national cancer statistics for the first phase. The number of new cases estimated for malignant tumors worldwide in 2014 is 380.4 ten thousand (211.4 ten thousand for men and 169.0 ten thousand for women), more than 1 thousand cases were diagnosed as cancer every day on average, and 7 cases were diagnosed as cancer every minute [1, 2 ]. At present, most malignant tumors have poor therapeutic effect, and one important reason is the heterogeneity of tumors. Tumor heterogeneity is the existence of multiple biological differences between different patients of the same tumor type or between different cells within the tumor of the same patient. This difference determines a significant difference in efficacy between patients with the same tumor for the same treatment regimen. At present, most doctors treat patients according to the standard scheme of the industry guidelines such as NCCN and the like, and the standard chemotherapy scheme is lack of corresponding pertinence for individuals and has certain blindness. Research shows that the average chemotherapy effective rate is only 25-30%, so that the medicine sensitivity experiment is utilized to guide personalized and accurate medicine application, the treatment effect is improved, and adverse drug reactions are reduced, and the research becomes a hot spot of domestic and foreign research.

The tumor microenvironment refers to a local internal environment formed by tumor cells, immune cells infiltrated locally by the tumor, stromal cells, endothelial cells, secreted active media and the like. Numerous experimental studies have demonstrated that a complex tumor microenvironment regulates tumor growth, infiltration and metastasis, and that tumor development is dependent on this complex dynamic microenvironment [3 ]. Some studies in recent years have shown that the surface tumor microenvironment is closely related to cell resistance. One important reason why the expected effects of drugs administered to cancer tumors are often not achieved is probably that healthy cells present in the tumor microenvironment provide the corresponding conditions for the cancer cells to resist the drug and survive. Changes the interaction between cancer cells and surrounding cells (tumor microenvironment), and can improve the efficacy of anticancer drugs [4 ].

Since tumor heterogeneity and microenvironment are closely related to drug response of tumors, an ideal method for tumor drug sensitivity detection needs to reproduce the heterogeneity and microenvironment of a patient's tumor. The human-derived tumor xenograft (PDX) model is a model of a graft tumor formed by directly implanting a tissue mass derived from a tumor Patient into an immunodeficient mouse. The PDX model preserves the original three-dimensional structure of tumor tissue, so that the heterogeneity and microenvironment of tumor are well restored in the mouse body, and the tumor drug sensitivity detection and clinical compliance rate by using the PDX model reaches about 90 percent, which is the most accurate drug screening method known at present [5 ]. However, the screening of tumor treatment schemes by using the PDX model requires a long time (generally 4-6 months), is high in cost (more than one hundred thousand), has high requirements on experimental conditions (an animal laboratory requiring SPF), and cannot well meet the actual clinical requirements.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a hydrogel-coated microtissue block culture drug sensitivity detection method.

In order to solve the technical problems, the invention adopts the following technical scheme: a hydrogel-coated microtissue block culture drug sensitivity detection method is characterized by comprising the following steps:

a) collecting a tumor specimen: cutting tumor tissue with vigorous growth and no ulcer necrosis at the edge of the tumor during operation, rapidly placing the tumor tissue into a centrifuge tube containing tissue preservation solution, and placing the centrifuge tube into an ice bag for low-temperature transportation;

b) tumor sample treatment: tumor tissue viability was detected in a laboratory biosafety cabinet with 0.4% trypan blue staining solution and non-viable tissue was removed. The tissue was then sterilized by soaking in 0.2% iodophor solution for 30 seconds and rinsed with 3% double antibody in PBS. Dividing the tumor tissue into 2X 2mm³The tissue block of (2) is placed in each hole of a 96-hole cell culture plate, a cut tissue block (cell culture plates or culture dishes with other specifications can be placed in a proper number of tissue blocks according to the volume size) is placed on ice or a blue ice brick for precooling;

c) tissue block inoculation: adding a certain amount of pre-cooled I type collagen/tissue culture medium mixed solution (80 ul, 96-hole cell culture plate) at 4 ℃ into cell culture holes containing tissue blocks, submerging all the tissue blocks, then covering the cell culture holes with a cover, putting the cell culture holes into a cell culture box with the carbon dioxide content of 37 ℃ and 5% for incubation for 2-3 hours, taking out the culture plate after the collagen is solidified, adding a proper amount of tissue culture medium (100 ul, 96-hole cell culture plate) into a biological safety cabinet, and then putting the culture plate into the cell culture box for culture.

d) And (3) drug treatment: after the tissue block is cultured in a cell culture box for 24 hours, the original culture medium is removed, a complete culture medium containing the test drug is added, and the complete culture medium containing the test drug is replaced every 24 hours;

e) and (3) sensitivity detection: after the tissue block is cultured for 3 to 7 days by using a complete culture medium medicine containing a test medicine, absorbing and removing the medicine-containing culture medium, washing the tissue block for 3 times by using a PBS (phosphate buffer solution), then adding an MTT (methyl thiazolyl tetrazolium) solution, absorbing the MTT solution after 6 hours, taking the tissue block out of the hydrogel by using an ophthalmic forceps, putting the tissue block into a new cell culture hole, adding 200ul of DMSO (96-hole cell culture plate) reagent, shaking for 10 minutes, absorbing 100ul of the solution from each hole of the cell culture plate filled with the tissue block, adding the absorbed solution into the hole plate of the new cell culture plate, and detecting the absorbance value of each hole by using a microplate reader;

f) and (4) analyzing results: and (4) converting the absorbance value of each hole of the pore plate according to an absorbance value conversion formula, and further calculating the influence of each drug on the activity of the tumor tissue mass.

Further, in step a), the collected tumor tissue is cut from a part with relatively vigorous tumor growth by a doctor during tumor resection, the resected tumor tissue is put into a 50ml centrifuge tube containing a tissue preservation solution, the specimen is transported to a laboratory in an ice pack at low temperature, and the time from the separation of the specimen to the low temperature transportation to the laboratory is not more than 48 hours at most, preferably within 2 hours.

Further, in step c), the adopted cell culture plate is a 96-hole, 48-hole or 24-hole plate, and the added type I collagen solution can be replaced by Matrigel gel or other temperature-sensitive hydrogel; during incubation, the pore plate is placed in a cell culture box, the temperature is controlled at 37 ℃, incubation is carried out for 2-3 hours, a proper amount of tissue culture medium is added into each pore after hydrogel is solidified, and then the culture plate is continuously placed in the cell culture box for culture.

Further, in step e), the MTT solution used may be replaced with a detection reagent such as CCK reagent, MTS reagent, or the like.

Compared with the prior art, the method can be called as a 'jelly' drug sensitive detection method, the method preserves the heterogeneity and microenvironment of tumors, is simple to operate, has low requirements on experimental conditions, can quickly and accurately screen out the tumor chemotherapy scheme most suitable for patients, and can also be used for detecting the response of normal tissues to various intervention measures, such as toxicology detection of normal liver tissues.

Detailed Description

The present invention will be described in detail with reference to examples.

The invention provides a hydrogel-coated microtissue block culture drug sensitivity detection method, which mainly comprises the following steps:

a. collecting a tumor specimen: tumor tissues which grow vigorously and are free from ulcer and necrosis are collected on an operating table by a doctor, and the tumor tissues are quickly put into a 50ml centrifugal tube containing tissue preservation solution and taken back to a laboratory within 2 hours by using an ice bag.

b. Tumor sample treatment: dividing the tumor tissue into 2X 2mm after the activity identification, trimming, disinfection and cleaning in a biological safety cabinet of a cell laboratory³The tissue block cell culture plate after cutting is placed in each hole of the 96-hole cell culture plate and is placed on ice or a blue ice brick for precooling.

c. Tissue block inoculation: adding a certain amount of pre-cooled I type collagen/tissue culture medium mixed solution (80 ul, 96-hole cell culture plate) with the temperature of 4 ℃ into cell culture holes containing tissue blocks, submerging all the tissue blocks, then covering the cell culture holes, putting the cell culture holes into a cell culture box with the carbon dioxide content of 37 ℃ and 5% for incubation for 2-3 hours, taking out the culture plate after the collagen is solidified, adding a proper amount of tissue culture medium (100 ul, 96-hole cell culture plate) into a biological safety cabinet, and then putting the culture plate into the cell culture box for culture.

d. And (3) drug treatment: the original culture medium was aspirated after 24 hours of tissue block culture, and the complete culture medium containing the test drug was added, and replaced every 24 hours.

e. And (3) sensitivity detection: after 3-7 days of administration, the drug-containing medium is aspirated and discarded, the solution is washed with PBS 3 times, then MTT solution (or detection reagents such as CCK and MTS) is added, after 6 hours, the MTT solution is aspirated, the tissue block is taken out from the hydrogel by using ophthalmological forceps, 200ul DMSO (96-well cell culture plate) reagent is added into a new cell culture hole and is vibrated for 10min, 100ul solution is aspirated from each hole, a new 96-well plate is added, and the absorbance value of each hole is detected on an enzyme-labeling instrument.

f. And (4) analyzing results: and (4) converting the absorbance value of each hole according to a formula, and calculating the influence of each drug on the activity of the tumor tissue mass. The formula is a = abc, where a is the absorption coefficient in L/(g · cm), b is the distance traveled by the light in the sample (typically the thickness of the cuvette) in cm, and c is the solution concentration in g/L.

In the test, the influence of each drug on the tumor tissue mass activity was judged with reference to the following table (table 1).

TABLE 1

Wherein, the drugs in table 1 are the detection results of the present invention, wherein the critical value of the inhibition rate is 30%, and the drugs with the inhibition rate lower than 30% can be regarded as ineffective drugs.

The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the patent and protection scope of the present invention should be subject to the appended claims.