CN114164250A - Individual drug sensitivity detection method for tumor tissue homogenate serum-replacement micro tissue block - Google Patents

Abstract

The invention provides a method for detecting individual drug sensitivity of tumor tissue homogenate serum-substituted micro tissue blocks, which belongs to the technical field of biological medicine and mainly comprises the following steps: preparing an artificial basement membrane three-dimensional tissue culture low-viscosity culture dish and a culture plate, processing the obtained fresh tumor tissue into tumor micro tissue blocks with uniform sizes, placing the tissue blocks into a low-adhesion plate containing a tissue fluid serum-substituted micro tissue block individualized culture medium for stable incubation, inoculating the stabilized tumor micro tissue blocks into the culture plate, and carrying out drug treatment and drug sensitive detection. The invention solves the problems of long time consumption and poor data correlation of screening of tumor personalized treatment schemes and evaluation of antitumor drugs, retains the heterogeneity and original microenvironment of tumors to the maximum extent, and ensures the efficacy of tumor tissues. The method is simple to operate and strong in feasibility, and the optimal individualized treatment scheme of the patient can be quickly and accurately obtained within 3 days.

Description

Individual drug sensitivity detection method for tumor tissue homogenate serum-replacement micro tissue block

Technical Field

The invention relates to the technical field of biomedicine, in particular to a tumor tissue liquid serum-substituted micro tissue block individualized in-vitro tumor tissue culture system, which can maintain the natural biological characteristics of tumor cells to the maximum extent in-vitro experiments, can quickly and accurately obtain drug sensitivity detection data and provide clinical basis for individualized and accurate treatment of tumor patients.

Background

In recent years, the incidence rate of malignant tumors is continuously increased, and the combination of surgery and chemotherapy is still the main means for treating tumors. However, due to differences between individuals, the sensitivity of the same type of tumor to different chemotherapeutic drugs varies. The in vitro chemotherapy drug sensitivity experiment can help oncologists to know the sensitivity of different patients to different chemotherapy drugs in advance, and make individualized chemotherapy schemes to realize accurate drug administration for tumor patients. Therefore, establishing a detection model which can keep the genomics and the genetics characteristics of parent tumor tissues in vitro, is simple to operate and easy to evaluate is an important way for improving the tumor chemotherapy effect and realizing the tumor individual treatment.

The organoids have the greatest advantages of being capable of being used as substitutes of tumors of patients, well keeping the heterogeneity of parent tumors, and simultaneously being capable of screening drugs in a high-throughput manner and accurately predicting the curative effect of anticancer drugs. The multicellular tumor spheroid model formed by cell aggregation is considered the "gold standard" for three-dimensional cancer models because of its simplicity and ease of use. The method is used for selecting the tumor tissue after the operation, establishing a tumor organoid culture system and an organoid drug screening platform based on a new technology of forming tumor three-dimensional microspheres by self-assembling primary tumor cells, and providing clinical basis for realizing personalized accurate treatment of tumor patients.

The existing anti-tumor drug sensitivity detection method has the following problems: 1. in vitro two-dimensional cell culture drug screening system, individuation is poor, the accuracy is not high, only 5% that can pass through clinical experiment finally, and easily produce resistant medicine, caused very big cost waste. 2. The establishment of an in vitro three-dimensional cell culture system can be carried out only when tumor tissues grow up to a certain degree, the period is long, the operation of the steps is complicated, the three-dimensional structure and the microenvironment of the in vivo tumor tissues cannot be accurately and individually reduced, and inconvenience is brought to clinical application.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide an in vitro tissue culture solution serum-substituted micro tissue block individualized tissue block culture system for anti-tumor drug sensitivity detection, a tissue sample is not required to be obtained through an operation, and large wound can not be caused to a patient; and the tumor early-stage detection can be carried out, the problems of long time consumption and poor data correlation of tumor personalized treatment scheme screening and antitumor drug evaluation are solved, the heterogeneity and the original microenvironment of the tumor are kept to the maximum extent, and the efficiency of tumor tissues is ensured.

The technical scheme adopted by the invention is as follows:

a tumor tissue homogenate serum-substituted micro tissue block individual drug sensitivity detection method comprises the following steps:

a) preparing an artificial basement membrane three-dimensional tissue in-vitro low-adhesion culture dish and a culture plate;

b) obtaining a fresh tumor tissue sample, and taking a part of the fresh tumor tissue sample to prepare tumor micro tissue blocks with uniform size;

c) weighing the rest part of the tissue blocks, transferring to a sterilized glass homogenizer, and preparing tissue homogenate to obtain a culture medium containing a certain proportion of tissue homogenate;

d) adding the tumor tissue blocks with uniform size in the step b) into a culture medium containing a certain proportion of tissue homogenate in the step c) to form a tissue homogenate serum-substituted micro tissue block individualized culture system at 37 ℃ and 5% CO₂Stably incubating the cell culture box with the content;

e) carrying out trypan blue activity detection on the tumor micro tissue block stabilized in the step d);

f) inoculating the active tumor micro tissue blocks into a low-adhesion culture plate, and incubating with chemotherapeutic drugs;

g) the glucose metabolism and MTT method are adopted to detect the drug sensitivity of the chemotherapy scheme.

Further, in step a), the prepared low-adhesion culture dish and culture plate is poly (2-hydroxyethyl methacrylate) (PHEMA) in order to prevent the cells in the tumor tissue block from migrating and growing adherently and maintain the natural tumor cell-matrix-cell interaction. The preparation method of the culture dish or the culture plate with the artificial basement membrane and the three-dimensional tissue in vitro low adhesion comprises the steps of adding PHEMA into 85% ethanol according to the ratio of 15mg:1mL, adding an ethanol solution of the polymer into the culture dish or the culture plate after the polymer is completely dissolved, drying a cell culture bottle at 37 ℃ for 24 hours, and preparing the culture dish or the culture plate with the artificial basement membrane and the three-dimensional tissue in vitro low adhesion for later use.

Further, in the step b), fresh tumor tissue is cut from a part with more vigorous tumor growth by a doctor during tumor resection operation, the resected tumor tissue is put into a 50ml centrifuge tube containing tissue preservation solution, the ice pack 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; washing the obtained fresh tumor tissue sample with PBS for 1-2 times, and preparing 1mm of active part³Tumor microtissue pieces of uniform size. Specifically, the preparation of the tumor tissue blocks with uniform size comprises the steps of obtaining a tumor tissue sample, placing a fresh tumor tissue block of a patient in a sterile container, adding PBS (phosphate buffer solution) for washing for 2 times, removing blood stains, necrosis, liquefaction, microorganism-contaminated tissues and the like, placing the fresh tumor tissue block in Hanks buffer solution for rapid cutting to obtain 1mm tumor tissue blocks³To a homogeneous tissue mass.

Further, in step c), the remaining portion of the tissue mass is weighed and transferred to a sterile glass homogenizer, following a mass: medium equal to 1 g: adding a culture medium in a proportion of 5mL, grinding, homogenizing, centrifuging, and collecting filtrate to obtain a tissue homogenate stock solution; diluting the filtrate and the serum-free culture medium according to different proportions to obtain tissue homogenate with the volume concentration of 10-20%, wherein the osmotic pressure can be influenced if the volume concentration is too high, and the environment in vivo cannot be simulated if the volume concentration is too low. Specifically, the culture medium is a serum-free culture medium; the centrifugation is carried out in a centrifuge tube under the conditions of 10000-.

Further, the stable incubation system in the step d) is that 3 tumor masses are added into each hole of a 96-hole plate, 200ul of the culture medium containing the tissue homogenate with a certain proportion is added to form a tissue homogenate serum-substituted microtissue block individualized culture system, and the cell culture box is stably incubated at 37 ℃ and the content of 5% CO 2.

Further, the method for detecting the activity of the microtissue blocks by the e) is trypan blue activity detection, the activity of tumor tissues is detected by 0.4 percent of trypan blue staining solution, tissues without activity are removed, and then the tissues are washed by PBS containing 3 percent of double antibodies;

further, in step f), the medium is separated for the next day, and the medium containing a proportion of the tissue homogenate is replaced every three days for a total of 5 days.

Further, in the step g), the glucose metabolism detection method comprises the steps of taking 20 mu L of supernatant from each hole of the inoculation hole to a culture plate, adding 80 mu L of o-toluidine solution, carrying out water bath in boiling water for 5min, and detecting the light absorption value by an enzyme-labeling instrument at 630 nm;

adding 20 mu L of MTT solution into each hole of an inoculation hole, incubating for 2-4h in an incubator until the color of the tissue becomes dark, discarding the supernatant, adding 150 mu L of DMSO into each hole, incubating for 30min in the incubator until the tissue becomes faded, taking the supernatant to a new 96-hole plate, and measuring the light absorption value at 540nm by using an enzyme-labeling instrument. The MTT solution can be replaced by CCK reagent, MTS reagent and other detection reagents.

And g) in the chemotherapy scheme sensitivity judgment step, converting the absorbance value of each hole of the hole plate, further calculating the influence of each drug on the activity of the tumor tissue block, and selecting a chemotherapy scheme with high activity inhibition rate and low glucose consumption rate by taking the activity inhibition rate as the main part and taking the glucose consumption rate as the auxiliary part.

Compared with the prior art, the invention has the beneficial effects that: (1) the homogenate of the tumor tissue prepared after tumor resection contains various cytokines, so that the uniformity of the homogenate and the in-vivo state is kept to the maximum extent in the in-vitro culture process, the in-vitro drug sensitivity experiment is closer to the real situation, the detection result is more reliable, and the screened drug is more effective in the body of a patient. Other methods mostly use non-self components such as bovine serum, compounds, synthetic proteins and the like, which may cause the change of the state of tumor cells, and cannot completely simulate the in vivo environment, so that the detection result may deviate from the actual situation. (2) The tumor tissue mass of the present invention is more realistic than "organ-like cells", which are grown from "stem cells", which are not real tumor fragments and take a long time to build up, and do not contain stromal cells because they are intuitively and rapidly available in response to anticancer drugs, because the extracellular matrix components in cancer and various stromal cells as well as tumors are not destroyed; (3) the method disclosed by the invention is simple to operate, has low requirements on experimental conditions, and can quickly and accurately screen out the tumor chemotherapy scheme most suitable for patients; the tumor tissue homogenate serum-substituted micro tissue block individual in-vitro culture system provided by the invention maintains heterogeneity and microenvironment of tumors to the maximum extent, and provides an in-vivo bionic drug screening platform for testing efficacy and toxic adverse reaction of candidate drugs. (4) The accurate and personalized tumor culture system can be applied to research and screening of drugs, research on drug efficacy and toxicity of the drugs, and research on sensitivity prediction and personalized treatment of chemotherapy, radiotherapy, targeted therapy and immunotherapy of clinical patients.

Drawings

FIG. 1: the method of the invention is a schematic flow chart; wherein, a, preparing tumor micro-tissues; b, tumor micro-tissue culture; c, drug stimulation; d, collecting data;

FIG. 2: after the drug treatment, the tumor micro tissue is cultured by tissue homogenate culture mediums with different proportions.

FIG. 3: MTT cell viability assay graph of tumor microtissue after drug treatment.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below. It is to be understood that the embodiments described are only a few embodiments of the present application and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Example 1:

transferring the tumor-bearing mice to a super clean bench after the tumor-bearing mice die at the cervical vertebra; taking a mouse tumor tissue block, transferring the mouse tumor tissue block into a culture dish, and washing the mouse tumor tissue block for 1-2 times by PBS (phosphate buffer solution); cutting the cleaned tissue block into pieces with high activity to a size of about 1mm³Uniform small blocks of (2); the remaining tissue mass was weighed, transferred to a sterile glass homogenizer, and expressed as tissue mass (g): serum-free medium (ml) equal to 1: 5, adding a serum-free culture medium according to the proportion, and grinding and homogenizing; transferring the grinded tissue homogenate liquid into a centrifuge tube, and centrifuging at 15000rpm for 5 min; centrifuging, transferring the supernatant into a new centrifuge tube, filtering through a 0.22um filter, and collecting filtrate; respectively mixing the filtrate and the serum-free culture medium according to different volume ratios of 1: 6 (15%), 1: 9 (10%), 1: 19 (5%) dilution to obtain a culture medium containing a certain proportion of tissue homogenate; adding 3 tumor masses into each hole of a 96-hole plate, making 200ul of the homogenate, making 5 multiple holes for each concentration, and setting a culture tumor tissue containing 10% autologous serum and 10% fetal calf serum as a control group; place 96-well plate in CO₂Incubation in an incubator; and (4) changing the culture solution the next day after separation, changing the culture solution every three days, and culturing for 5 days to obtain the tumor organoids.

Example 2:

tumor organoids obtained as described in example 1 were used as susceptibility tests for vincristine, epirubicin, pirarubicin, docetaxel, 5-fluorouracil, cisplatin, cyclophosphamide, gemcitabine and oxaliplatin. After 3 days of administration, 20. mu.L of MTT solution at 5mg/ml per well was added, incubation was continued for 4 hours, the culture was terminated, the medium was aspirated, 150. mu.L of DMSO per well was added, shaking was carried out for 10min, the wavelength was chosen at 490nm, and the absorbance of each well was measured on an ELISA detector.

The inhibition rate of cell activity is (control group absorbance-experiment group absorbance)/control group absorbance is 100%, and when the inhibition rate is more than 30%, the medicine is effective.

As can be seen from fig. 2, when tumor tissues were cultured in 15%, 10%, and 5% tumor tissue homogenates, 10% autologous serum, and 10% fetal bovine serum, respectively, the migration ability of tumor cells was found to be significantly higher in the 15% and 10% tumor tissue homogenates groups than in the other groups, indicating that the tumor tissue homogenates could better maintain the characteristics of tumor cells.

FIG. 3 is a graph showing the results of sensitivity of tumor microtissue blocks to various chemotherapeutic drugs, wherein the threshold of inhibition is 30% and drugs with inhibition less than 30% are considered ineffective.

Claims (10)

1. A tumor tissue homogenate serum-substituted micro tissue block individual drug sensitivity detection method is characterized by comprising the following steps:

a) preparing an artificial basement membrane three-dimensional tissue in-vitro low-adhesion culture dish and a culture plate;

b) obtaining a fresh tumor tissue sample, and taking a part of the fresh tumor tissue sample to prepare tumor micro tissue blocks with uniform size;

c) weighing the rest part of the tissue blocks, transferring to a sterilized glass homogenizer, and preparing tissue homogenate to obtain a culture medium containing a certain proportion of tissue homogenate;

d) adding the tumor tissue blocks with uniform size in the step b) into a culture medium containing a certain proportion of tissue homogenate in the step c) to form a tissue homogenate serum-substituted micro tissue block individualized culture system at 37 ℃ and 5% CO₂Stably incubating the cell culture box with the content;

e) carrying out trypan blue activity detection on the tumor micro tissue block stabilized in the step d);

f) inoculating the active tumor micro tissue blocks into a low-adhesion culture plate, and incubating with chemotherapeutic drugs;

g) the glucose metabolism and MTT method are adopted to detect the drug sensitivity of the chemotherapy scheme.

2. The method for individually and sensitively detecting the tumor tissue homogenate serum-substituted micro tissue block according to claim 1, wherein the low-adhesion culture dish or culture plate in the step a) is poly (2-hydroxyethyl methacrylate), and the method comprises the specific steps of adding PHEMA into 85% ethanol according to a ratio of 15mg:1mL, adding an ethanol solution of the polymer into the culture dish or culture plate after the polymer is completely dissolved, drying the cell culture flask at 37 ℃ for 24 hours, and preparing an artificial basement membrane three-dimensional tissue in vitro low-adhesion culture dish or culture plate for later use.

3. The method of claim 1, wherein the tumor tissue homogenate serum-substituted micro tissue block individualized susceptibility testing is performed by preparing a tumor micro tissue block with uniform size in step b), obtaining a tumor tissue sample, placing a fresh tumor tissue block of a patient in a sterile container, adding PBS for washing for 2 times, removing blood stain, necrosis, liquefaction and microorganism contamination tissues, placing the fresh tumor tissue block in Hanks buffer solution for rapid cutting to obtain 1mm tumor tissue homogenate serum-substituted micro tissue block individualized susceptibility testing³To a homogeneous tissue mass.

4. The method of claim 1, wherein the tissue homogenate of the step c) is prepared by weighing the rest of the tissue homogenate, transferring the weighed tissue homogenate to a sterilized glass homogenizer, and performing individual drug sensitive detection on the tissue homogenate by: serum-free medium equal to 1 g: adding a serum-free culture medium in a proportion of 5mL, and grinding and homogenizing; transferring the grinded tissue homogenate liquid into a centrifuge tube, and centrifuging at 15000rpm for 5 min; centrifuging, transferring the supernatant into a new centrifuge tube, filtering through a 0.22um filter, and collecting filtrate as a tissue homogenate liquid stock solution; diluting the filtrate and the serum-free culture medium according to different proportions to obtain tissue homogenate with the volume concentration of 10-20%.

5. The method of claim 1, wherein the stable incubation system in step d) is to add 3 tumor mass per well in a 96-well plate, 200ul of the culture medium containing a certain proportion of tissue homogenate to form a tissue homogenate serum-replaced micro tissue mass individualized culture system, and incubate the tissue homogenate serum-replaced micro tissue mass in a cell culture box with 5% CO2 at 37 ℃.

6. The method of claim 1, wherein the detection of the activity of the microtissue blocks by e) is trypan blue activity detection, 0.4% trypan blue staining solution is used to detect the activity of the tumor tissue, the tissue without activity is removed, and the tissue is washed with 3% double-antibody-containing PBS.

7. The method according to claim 1, wherein the step f) comprises incubating with chemotherapeutic drugs in a blank control well and a drug treatment well at 37 deg.C and 5% CO₂The incubation time of the cell incubator was 72 h.

8. The method of claim 1, wherein in the step g), 20 μ L of the supernatant is collected from each well of the inoculation hole and added to a culture plate, 80 μ L of o-toluidine solution is added, the culture plate is bathed in boiling water for 5min, and the absorbance is detected by a microplate reader at 630 nm.

9. The method for detecting the individual susceptibility of the tumor tissue homogenate serum-substituted micro tissue block according to claim 1, wherein in the step g), 20 μ L of MTT solution is added into each inoculation hole, the culture box is incubated for 2-4h, when the color of the tissue becomes dark, the supernatant is discarded, 150 μ L of DMSO is added into each inoculation hole, the culture box is incubated for 30min, when the tissue becomes dark, the supernatant is taken out to a new 96-well plate, and the absorbance value at 540nm is measured by a microplate reader.

10. The method of claim 1, wherein the sensitivity of the chemotherapy regimen in step g) is determined by converting the absorbance of each well of the well plate, and calculating the effect of each drug on the activity of the tumor tissue mass, and a chemotherapy regimen with high inhibition of activity and low glucose consumption is selected with the inhibition of activity as the main component and the glucose consumption as the auxiliary component.

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