CN117004572A - Construction method and application of patient-derived transplanted tumor organoid model PDXO - Google Patents
Construction method and application of patient-derived transplanted tumor organoid model PDXO Download PDFInfo
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- CN117004572A CN117004572A CN202311009790.5A CN202311009790A CN117004572A CN 117004572 A CN117004572 A CN 117004572A CN 202311009790 A CN202311009790 A CN 202311009790A CN 117004572 A CN117004572 A CN 117004572A
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Abstract
The invention relates to a construction method and application of a patient-derived transplanted tumor organoid model PDXO. The construction method comprises the following steps: obtaining a patient-derived transplanted tumor organoid model PDXO by using a PDX model tumor tissue block through a tissue direct culture method or a cell culture amplification method; the tissue direct culture process comprises the following steps: placing tumor tissue fragments in a organoid culture medium for culture or embedding the tissue fragments cut by the tumor tissue blocks into matrigel gel drops and immersing the matrigel drops in the organoid culture medium for subculture to obtain a patient-derived transplanted tumor organoid model PDXO; the cell culture expansion method comprises the following steps: and (3) digesting the tumor tissue block into single cells or cell clusters with tumor dryness, re-suspending the single cells or cell clusters in a Matrigel culture medium, and then carrying out Matrigel dome gum titration, and culturing, liquid exchange and passage to obtain the patient-derived transplanted tumor organoid model PDXO.
Description
Technical Field
The invention belongs to the field of biological cell/tissue culture, and particularly relates to a construction method and application of a patient-derived transplanted tumor organoid model PDXO.
Background
Experimental models are critical for disease research. Traditionally, preclinical pharmacodynamic studies of cancer have relied primarily on in vitro cultured cell/tissue models and mouse engraftment tumor models inoculated with human tumor tissue. The in vitro cultured cell/tissue model is an experimental model obtained by taking an established tumor cell line capable of being subjected to unlimited division propagation as a material and carrying out in vitro culture in a corresponding culture container and culture medium system, and is used for researching the pathogenesis of cancer and utilizing the cancer pathogenesis and the tumor cell/tissue model to research and evaluate the effectiveness of different diagnosis and treatment means. The mouse transplantation tumor model inoculated with human tumor tissue takes an immunodeficiency mouse as a carrier, and human cancer cells (namely CDX model) or tumor tissue blocks (namely PDX model) from patients are grafted into the mouse body for culture, so that a corresponding cancer experimental model is obtained. The model can help us analyze and evaluate the effectiveness and prognosis of different diagnosis and treatment means. With the development of technology, the further humanized reconstruction of the mouse model can be realized by further technologies such as gene editing reconstruction and the like, so that the real situation in a patient can be better simulated.
Cancer is a highly heterogeneous, highly personalized systemic disease. With the penetration of cancer research and strategies, requirements of "refinement", "individualization", "higher clinical relevance", etc. are put forward and emphasized, which put higher demands on experimental models related to cancer. For in vitro experimental models based on cell/tissue culture techniques, the number of cell lines currently established and in commercial use is only about a few thousand, while the success rate of transforming patient-derived tumor tissue into cell lines that can proliferate in an unlimited division is low. Although different culture or engineering techniques (e.g., 2D/3D culture, resistance induction, cell fusion, genetic engineering, etc.) can be currently employed to further enrich the size of the variety and number of in vitro cell lines and to increase their clinical relevance, such number scales are far from those of cancer patients worldwide. Specifically, the richness of the scale, clinical relevance indexes and the like still need to be improved so as to achieve accurate description of the real disease characteristics of the patient by the experimental model, and therefore more accurate data results are obtained to guide subsequent research and development. On the other hand, in-vivo experiments based on CDX and PDX animal models can compensate the limitation of in-vitro experimental models to a certain extent, provide higher clinical relevance and more realistic simulation of diseases, but still cannot completely replace in-vitro experiments due to the problems of long model building period, high cost, inapplicability to rapid high-throughput screening experiments and the like.
Disclosure of Invention
The invention aims to provide a construction method and application of a patient-derived transplanted tumor organoid model PDXO.
In a first aspect, the present invention provides a method for constructing a patient-derived transplantation tumor organoid model PDXO, the method comprising: extracting and separating tumor tissue blocks from the PDX model, and obtaining a patient-derived transplanted tumor organoid model PDXO by using the tissue blocks through a tissue direct culture method or a cell culture amplification method;
the process of the tissue direct culture method comprises the following steps: cutting a tumor tissue block into tissue fragments and placing the tissue fragments in an organoid culture medium for culture, or embedding the tissue fragments cut by the tumor tissue block into matrigel gel drops and immersing the matrigel drops in the organoid culture medium for subculture to obtain a patient-derived transplanted tumor organoid model PDXO;
the cell culture expansion method comprises the following steps: and digesting the tumor tissue block into single cells or cell clusters with tumor dryness, re-suspending the single cells or cell clusters in a Matrigel culture medium, dripping Matrigel suspension into an orifice plate for incubation, dripping Matrigel dome glue for shaping, and culturing, changing liquid and passaging to obtain the patient-derived transplanted tumor organoid model PDXO.
Preferably, the organoid medium comprises the components: duplex modified Eagle Medium/nutrient mixture F-12, glutamine derivative (1 x), B27 (1 x), N2 (1 x), penicillin/streptomycin (1 x), primocin, noggin, EGF, wnt a, R-spondins, A83-01, Y-27632, SB202190, N-acetyl-L-cysteine, nicotinamide.
Preferably, in the process of the tissue direct culture method, the dosage ratio of the tumor tissue mass to the organoid culture medium is controlled to be 0.1-1.0g:5-50mL.
Preferably, in the process of the tissue direct culture method, the culture algebra is more than 10 generations.
Preferably, in the process of the cell culture amplification method, collagenase and TrypLE are adopted to digest tumor tissue blocks in two steps to obtain single cells or cell clusters with tumor stem property.
Preferably, the two-step digestion process of the tumor tissue blocks by collagenase and TrypLE comprises the following steps: mixing the tumor tissue block with collagenase digestive juice for digestion, stopping digestion until all cells are observed to exist in a single cell or multicellular cluster form and the mixed solution becomes turbid, centrifuging, and then adding TrypLE digestive juice into the centrifuged precipitate for digestion;
the collagenase digestive juice component comprises: collaganaseIV with activity >250U/mL, DNase I with activity >250U/mL, 10mu. M Y-27632, DMEM/F12 medium, penicillin-streptomycin (1X).
Preferably, the cell concentration of the Matrigel suspension droplets is controlled to be 20,000-100,000 viable cells/. Mu.L.
Preferably, in the process of the cell culture expansion method, the liquid is changed every 2-4 days, and the passage is carried out every 7-10 days.
In a second aspect, the present invention provides a patient-derived transplanted tumor organoid model PDXO obtained according to the above construction method.
In a third aspect, the invention provides an application of the patient-derived transplanted tumor organoid model PDXO in high-throughput drug sensitive screening, PDXOX inoculation, in-vitro immune killing experiments and pathological sample preparation.
Advantageous effects
Compared with the traditional cell/tissue culture, the PDXO technology provided by the invention can provide a new in-vitro experimental model, has higher clinical relevance, better reserves the biological markers and pathological features in tumor tissues of patients, and is suitable for high-throughput screening experimental design. In the drug research and development and preclinical research stage, the PDXO model can be utilized to carry out in-vitro drug effect test, test and evaluation are carried out on the curative effect of the tested object, more accurate data guidance is provided for the in-vivo test of the subsequent PDX animal model, and the accuracy, timeliness and cost performance of the research are further improved;
compared with an animal model, the PDXO in-vitro experimental model provided by the invention has the advantages of relatively short experimental period, relatively low cost, suitability for high-throughput screening and the like, further enriches experimental models for cancer research, becomes another experimental model option for bridging in-vitro experiments and in-vivo animal experiments, and provides more accurate, clinically more relevant, cost-effective and timeliness-effective experimental results for cancer research;
the PDXO model provided by the invention can be used for carrying out expansion culture on a PDX model which grows slowly or is not easy to form tumors, and provides a new option for PDX expansion and library establishment.
Drawings
FIG. 1 is a schematic flow diagram of a method for constructing a patient-derived transplanted tumor organoid model PDXO;
FIG. 2 is a line graph of the bright field photograph and size change with time of culture of human colon cancer transplantable tumor PDXO prepared in example 1;
fig. 3 is a photograph of four different colon cancer PDXOs constructed in accordance with the present invention;
FIG. 4 is a photograph of a bright field of a intestinal organoid constructed in accordance with the present invention and a photograph of a HE pathological tissue;
FIG. 5 is a photograph of the bright field of a human colon carcinoma graft PDXO prepared according to the present invention (top) transferred into a 96 well plate and its bright field after 72 hours of co-incubation with Irinotecan (Irinotecan) and Fluorouracil (5-Fluorouracil) at different concentrations;
FIG. 6 is a graph comparing the results of quantitative analyses of the sensitivity of the human colon carcinoma graft PDXO shown in FIG. 5 to Irinotecan (Irinotecan) and Fluorouracil (5-Fluorouracil) using two methods, bright field photography (BF) and ATP-chemiluminescent cell viability assay kit (CellTiterGlo or CTG);
FIG. 7 is a graph showing the results of a test on the PDX model of human colon cancer transplantable tumor animals of the same origin as FIG. 6 for both Irinotecan (Irinotecan) and Fluorouracil (5-Fluorouracil, 5-Fu).
Detailed Description
The present invention is further illustrated by the following embodiments, which are to be understood as merely illustrative of the invention and not limiting thereof.
The invention utilizes the organoid culture technology to culture the tumor tissue blocks (namely PDX model) of the patient in vitro, and establishes a corresponding organoid in vitro model (PDXO). Compared with the traditional in vitro cell/tissue experimental model, the PDXO model has extremely high clinical relevance, and can highly reduce pathological characteristics such as heterogeneity, gene phenotype, biological properties, biomarkers and the like in the interior of tumors (and in patients) on the PDX model in vitro; compared with an animal model, the PDXO model has the advantages of relatively short experimental period, relatively low cost, suitability for high-throughput screening and the like, further enriches experimental models for cancer research, becomes another experimental model option for bridging in-vitro experiments and in-vivo animal experiments, and provides more accurate, clinically relevant and cost-performance and timeliness-higher experimental results for cancer research. In addition, the in vitro amplified PDXO tissue can be re-inoculated on a mouse to establish a PDXOX animal model, so that a new option with higher convenience and cost performance is provided for the amplification of a PDX sample library, and the PDXO tissue can be better complemented with an animal experiment model to jointly advance.
The basis of PDXO organoids is a patient-derived tumor tissue mass PDX model. The following is an exemplary description of a method for constructing a patient-derived transplantation tumor organoid model PDXO according to the present invention, with reference to fig. 1. The construction method of the patient-derived transplanted tumor organoid model PDXO comprises the following steps: extracting and separating healthy transplanted tumor tissue blocks from a PDX model mouse, cutting the tumor tissue blocks, storing the tumor tissue blocks in a fresh culture medium added with double antibodies, and storing and transporting the tumor tissue blocks on ice or in a refrigerator at a temperature of between 2 and 8 ℃; and then, the sheared freshly extracted tumor tissue blocks are subjected to a tissue direct culture method or a cell culture amplification method within 24 hours to obtain the patient-derived transplanted tumor organoid model PDXO.
The freshly extracted tissue blocks can be cut into small parts to prepare quick freezing samples and pathological samples for gene expression and pathological feature analysis. The establishment of the PDXO model requires the use of fresh PDX tissue, namely the tissue should be treated within 24 hours after the PDX tissue is obtained from the animal model, so that the apoptosis of the PDX tissue can be avoided from happening too long in vitro.
In some embodiments, the tissue direct culture method may employ the following process: cutting the tumor tissue block into tissue fragments or fragments with the diameter of 50-100 mu m, placing the tissue fragments or fragments into a culture dish, adding an organoid culture medium prepared by corresponding cancer seeds, and culturing on an experimental plate shaking table in a cell incubator; according to the tumor type, when the diameter size of the spherical tissue block to be grown normally exceeds 100 mu m (for example, the diameter size is 100-500 mu m), further cutting the spherical tissue block into fragments with the diameter of 100 mu m, and culturing the spherical tissue block in a liquid-changing mode; or embedding the tissue fragments or fragments into matrix gel drops, immersing the tissue fragments or fragments into a organoid culture medium together for subculture, and obtaining the patient-derived transplantation tumor organoid model PDXO.
In some embodiments, the organoid medium is designed and formulated separately for different cancer species and different tissues. The components of the organoid medium may include: duplex medium/nutrient mixture F-12 (advanced DMEM/F12), glutamine derivatives (Glutamax) (1 x), B27 (1 x), N2 (1 x), penicillin/Streptomycin (Penicillin-Streptomycin) (1 x), 0.1mg/mL Primocin, 100ng/mL Noggin, 50ng/mL EGF, 100ng/mL Wnt3a, 250-1000ng/mL R-spondins, 500nM A83-01, 10 μ M Y-27632, 3-10 μM SB202190, 1-1.25mM N-Acetyl-L-Cysteine (N-Acetyl-L-Cysteine), 10mM Nicotinamide (Nicotinamide).
Wherein, glutaMax, B27 and N2 are nutritional agents added in a supplementing way, penicillin-Streptomycin and Primocin are antibiotics for inhibiting the growth of fungi/bacteria/mycoplasma, noggin, EGF, R-spondins and Wnt3a are growth factors which are necessary for differentiating stem cells in organoids, and A83-01, SB202190, Y-27632, N-Acetyl-L-Cysteine and Nicotinamide mainly play roles in helping to regulate cell growth signal paths, provide oxidation resistance protection, inhibit apoptosis, inhibit the growth of impurity cells, promote activation of stem cells and the like. The organoid culture medium with the composition provided by the invention can ensure that tumor tissues from patients normally grow in vitro.
As an example, the media composition of the construction of the intestinal organoid model PDXO may include: advanced DMEM/F12, glutaMax (1 x), B27 (1 x), N2 (1 x), penicillin-Streptomycin (1 x), 0.1mg/mL Primocin, 100ng/mL Noggin, 50ng/mL EGF, 100ng/mL Wnt3a, 500ng/mL R-spondins, 500nM A83-01, 10 μ M Y-27632, 3 μM SB202190, 1mM N-Acetyl-L-Cysteine, 10mM Nicotinamide, gamin, 10ng/mL FGF-2, 20 ng/mLFG-10, 1 μMPGE2. Compared with basic culture medium components, the intestinal cancer organoid culture medium is also added with three growth factors of Gastin, FGF-2 and FGF-10 and PGE2 prostaglandin.
In some embodiments, the tumor tissue mass to organoid medium ratio can be 0.1-1.0g:5-50mL.
The temperature of the cell incubator may be 37 ℃, the humidity > 60%, and the carbon dioxide concentration may be 5vol%. The flat table shaking table can adopt a form of shaking at a low speed of 50-75 rpm.
In general, the tissue mass will become uniformly spherical in shape and gradually larger under normal growth conditions. The organoid culture process is static tissue culture, and nutrient substances in the culture medium are conveyed to cells and tissue positions in a free diffusion mode. In the culturing process, the size of the 3D structure organoid tumor tissue can be controlled within the range of 50-500 mu m, preferably 100 mu m, and the organoid within the size range can ensure that the in vitro culture of the PDXO tissue maintains the characteristics of heterogeneity, hypoxia center and the like in the approximation body, and can ensure that the PDXO tissue maintains growth without excessive apoptosis or degeneration caused by oversized tissue and incapacity of taking nutrition by internal cells. In addition, for determining the genetic stability of organoid tumor tissue, whole exon DNA sequencing and transcriptome sequencing and pathology studies can be performed, and the results can be compared with the original PDX tissue to verify the genetic stability characteristics.
In some embodiments, the number of liquid change incubations may be 5-10.
In some embodiments, the tumor tissue mass to matrigel droplet may be used in an amount of 0.05-0.5 g:200-2000 μl.
The matrix gel may be selected from Matrigel matrix gels. When Matrigel dome glue drops are used for culturing, the contact combination of polystyrene materials on the inner wall of a culture dish cannot simulate the real environment in vivo, so that the biological characteristic expression of tumor tissues can be changed, and the cells are ensured not to be in adherent contact with the inner wall of a culture container by Matrigel embedding or tissue mass passage. In some embodiments, the Matrigel may use a reduced growth factor (growth factor reduced) with no Phenol red (Phenol red free type) to reduce unwanted interference and effects of growth factor and Phenol red (Phenol red) on tissue growth.
In some embodiments, the tissue direct culture method may provide a culture algebra of the patient-derived transplanted tumor organoid model PDXO of >10 passages.
The tissue direct culture method disclosed by the invention takes tissue blocks with the size of hundreds of micrometers as culture starting materials, can furthest reserve the characteristics of cases in solid tumor tissues, such as heterogeneity in tumors, high osmotic pressure in tumors, hypoxia environment, substance exchange gradient and the like, and has the characteristics and advantages that the tissue obtained by final culture is spherical, opaque and compact in structure, which are not possessed by a tumor organoid model obtained by taking single cells or cell clusters as starting points and relying on a 3D cell culture technology. The model method provided by the invention has higher clinical relevance to the simulation of partial cancer species (such as brain glioma).
In the invention, the cryopreservation method of the prepared PDXO tissue spheres can be similar to the cryopreservation method of the PDX tissue spheres. The method specifically comprises the following steps: dispersing tissue balls with the diameter of 100 mu m in frozen stock solution at the concentration of 10-30 grains/mL per tube; and (3) using a program cooling box to carry out gradient cooling to-80 ℃, and then freezing into liquid nitrogen for long-term storage. And (5) preserving the basic identity information such as the name and source of the time scale tissue, the establishment date of the PDXO, the freeze thawing times, the culture algebra during freezing, the freezing date and the like. Thawing tissue according to normal method during resuscitation, and culturing after placing into culture medium.
The formula of the frozen stock solution can be as follows: 10vol.% dimethyl sulfoxide (DMSO), 20-40vol.% Fetal Bovine Serum (FBS) or reconstituted cell culture frozen stock (Recovery Cell Culture Freezing Medium), 50-70vol.% complete medium. The basic component of the complete culture medium is a DMEM/F12 culture medium; meanwhile, according to different cancer types, the nutrient components such as rh-Noggin, rh-RSPO1, rh-EGF, L-Wnt-3A conditioned medium, Y-27632, A83-01, SB202190 and the like with corresponding concentrations can be properly selected and added.
The tissue direct culture method provided by the invention is suitable for the scenes of organoid preparation of partial cancer species, establishment of a PDXOX model, qualitative research of in-vitro immunotherapy drug effect, biomarker and pathological feature research and the like.
In some embodiments, the cell culture expansion method may employ the following process: cutting the tumor tissue block into tissue fragments as small as possible, re-suspending the tissue fragments in fresh organoid culture medium added with the diabody, stirring, standing and centrifuging to obtain supernatant cell suspension; then, adopting collagenase and TrypLE to digest residual tissue fragments in two steps to obtain single cells or cell clusters with tumor stem property, filtering the single cells or cell clusters by a 70-100 mu m screen, centrifuging the cell filtrate filtered by the screen, removing supernatant, and re-suspending the obtained single cells or cell clusters with tumor stem property in 20-30mL of PBS; flocculent or fibrous sediment (generated by the rubber-linked agglomeration of non-tumor cell small-size residual impurities) observed in the resuspension process is removed by using a pipette, then the cells are counted, and data (cell activity > 30%) related to the cell concentration and the activity are collected; according to the actual cell number, sucking the cell suspension with the total number of living cells not exceeding 1.5X10-8, centrifuging, removing the supernatant, placing the centrifuge tube on ice for cooling, and then precipitating and re-suspending in Matrigel culture medium with the concentration of > 50wt%; and then dripping Matrigel suspension into corresponding holes of the pore plate, transferring the Matrigel suspension into a cell culture box, incubating for 10-30 minutes, completing Matrigel dome glue dripping and shaping, adding a culture medium for culturing, and performing liquid exchange and passage to obtain the patient-derived transplantation tumor organoid model PDXO.
In some embodiments, the process of stirring, standing, centrifuging to obtain a supernatant cell suspension may be: transferring the double-antibody culture medium containing tissue fragments into a centrifuge tube, standing for 30 seconds for layering, settling massive tissue fragments to the bottom of the tube, sucking the supernatant cell suspension, only keeping enough residual liquid which is not enough to be used for the tissue fragments, and sieving the sucked supernatant cell suspension through a 70 mu m filter screen to filter the tissue or fiber fragments with larger size, and only keeping single cells and cell clusters; transferring the filtrate into another centrifuge tube for preservation; then, adding a proper amount of culture medium into the residual tissue blocks precipitated at the bottom of the tube for rinsing, repeating the operations of standing, extracting the supernatant and sieving for 1 time, and enriching the filtered supernatant; centrifuging at 4deg.C and 1200rpm for 5 min, removing supernatant, counting cells, and storing on ice to obtain supernatant cell suspension.
In some embodiments, the process of two-step digestion of residual tissue fragments with collagenase, trypLE, may be: transferring the residual tissue fragments which are separated from the supernatant after standing and settled at the bottom of the centrifuge tube into a 6-hole or 12-hole plate, adding collagenase digestive juice, and putting into a cell incubator for digestion, wherein the collagenase dosage can be controlled to be 0.1-1.0 cm each time 3 The PDX tissue consumes 1-5 mL of digestive juice, and too little digestive juice can cause low digestion efficiency and too much digestive juice can cause waste; then, the porous plate is put into contact withShaking at 60-100rpm at 37deg.C for 0.5-24 hr, homogenizing cell tissue digestive juice every 10-15 min, observing cell tissue morphology with microscope until all cells are observed under microscope to exist in single cell or multicellular cluster form and the solution becomes turbid (if there is no shaking table, digestion can be performed in 37 deg.C incubator, plate is removed every 10-15 min, and blown 20 times with pipetting gun, and observed under microscope); digestion was terminated by dilution with an equal amount of DMEM/F12 medium or PBS; after stopping digestion, transferring the cell suspension into a centrifuge tube and standing, transferring the supernatant containing cells and cell clusters into another centrifuge tube and storing at 4 ℃ for standby (without sucking large-size tissue fragment sediment, leaving a small amount of liquid just covered with tissue fragments); then, 1-2mLTrypLE digestive juice is added into the tissue sediment after centrifugation and transferred to a cell culture box for digestion treatment, and after digestion, the tissue sediment is used>Dilution with 2ml pbs terminated digestion.
In some embodiments, the collagenase digestive juice is a universal digestive juice, and the components thereof may include: collagenase IV with activity >250U/mL, DNaseI with activity >250U/mL, 10. Mu. M Y-27632, DMEM/F12 medium, penicillin-streptomyin (1X). When the PDX tissue is digested to obtain single cells or cell clusters, the digestive juice used for tissue digestion contains a collaganase IV solution with activity higher than 250U/mL, and the digestive juice is suitable for decomposing collagen network structures in various tumor tissues and releasing the cells; in addition, DNaseI of more than 250U/mL is added, so that the DNaseI can be used for degrading NET released by neutrophils and DNA substances released by dead cells to help the release of the cells; in addition, Y-27632 and diabody are added into the digestive juice, so that the purposes of reducing apoptosis in the digestive process and reducing the risk of infecting microorganisms by cell tissues in the operation process can be achieved.
In some embodiments, the cell concentration of the Matrigel suspension droplets can be controlled to be 20,000-100,000livingcells/μl, and the well plate can be selected from room temperature microwell plates (conventional TC microwell plates). The amount of Matrigel suspension droplets can be controlled as follows: 1 drop of glue for each well of the 6-well plate, 50 mu L of glue for each well of the 12-well plate, 25 mu L of glue for each well of the 12-well plate, and 1 drop of glue for each well of the 24-well plate, and 10 mu L of glue for each well of the 24-well plate. If the volume of each glue drop is less than 30 mu L, the inverted micro-pore plate can be turned over carefully, and cells are enriched in the matrigel by utilizing the gravity action, so that the cell sedimentation is avoided and the bottom surface of the micro-pore plate is contacted.
In some embodiments, the temperature of the cell incubator may be 37 ℃ and the incubation time may be 10 to 30 minutes to solidify and set the matrigel.
In some embodiments, the fluid change is performed every 2-4 days and the passage is performed every 7-10 days. The liquid exchange and passage frequency can well maintain the shape and size change (50-500 mu m) of the tissue, avoid overgrowth of the tissue, and break away from the logarithmic growth phase to lose the healthy division function. After Matrigel is solidified and coagulated, the time for maintaining the complete structure under the environmental condition of the culture medium is generally about 14 days, and the time for subculturing the organoids can be well matched.
When the liquid exchanging operation is carried out, the micro-pore plate can be inclined, and the solution is sucked out from the edge of each hole; then, a culture medium is added along the side wall of each hole, so that the disturbance of matrigel drops is avoided in the whole process.
When the passage operation is carried out, the original culture medium is removed firstly, then cold PBS is used for rinsing, and the glue drops are cooled; after removal, using a cell recovery solution (Cell Recovery Solution) or a mild cell dispersion solution (Gentle Cell Dissociation Solution) to blow off glue drops, then incubating for 10-15 minutes at 4 ℃, degrading and destroying Matrigel structure, releasing organoid cells/cell spheres into the solution, adding PBS to dilute the cell recovery solution, and centrifugally cleaning for 2-4 times, wherein the centrifugal speed is 1200-1500rpm each time, and the temperature is 4 ℃ for 5 minutes; when removing the supernatant after centrifugation, a pipette is used for sucking away as much supernatant as possible, so that the loss caused by disturbing the cell mass which is enriched at the bottom of the centrifuge tube and is wrapped by the residual matrigel is avoided. Cell counting is carried out after re-suspending in a proper amount of culture medium, the re-suspending is carried out and then dispersed in a proper amount of mixed solution of the culture medium and Matrigel (ice operation), the cell concentration in the final mixture is in the range of 500-5,000 cells/mu L, matrigel dome glue drops are manufactured according to the method established by the model above, and culturing is carried out.
The organoid passaging operation involves breaking the Matrigel structure, which can release the 3D cell/tissue structure embedded therein, by physical means of mechanical force, i.e. by rinsing with cold PBS and blowing with a pipette to break Matrigel; preferably, the Matrigel structure is degraded using Cell Recovery Solution produced by Corning, gentle Cell Dissociation Reagent produced by stemcel, or a self-formulated Dispase I digestion solution to release cells.
The cryopreservation method of PDXO cells is similar to the cryopreservation method. In some embodiments, the organoids with >2 passages that are healthy and healthy may be collected 3-4 days from their last passage (more than 2 passages, ensuring that organoids are stable in shape, and the purpose of collection 3-4 days after the last passage is to ensure that more cells are in logarithmic growth phase, with 3D tissue architecture around 100 μm). After cell counting, the cells are resuspended in the frozen stock solution, and 1-5million cells per tube are dispersed in a proper amount of frozen stock solution (such as 1mL per tube); the program cooling box is used, after the temperature is cooled to-80 ℃ in a gradient way, the program cooling box is frozen into liquid nitrogen for long-term storage, when the program cooling box is stored, the basic identity information such as the name and the source of a tissue, the establishment date of a PDXO, the freezing and thawing times, the culture algebra during freezing and the freezing and thawing date and the like are marked, when the program cooling box is used for resuscitation, the program cooling box is basically similar to normal cell resuscitation operation, and after cell counting, the program cooling box is dispersed into a mixed solution of Matrigel and a culture medium for preparing glue drops for culture.
The counting of the organoids involves counting the cell spheres of the 3D structure, the collection of organoids ensures that the cells are in a vigorous logarithmic phase, and the diameter of the 3D tissue structure can be controlled to be about 50-200 μm. Thus, the time points for passaging and collection of organoids (e.g., the third day after passaging) should be well known at the time of operation, and the organoids should be screened using a 70 μm screen and counted using a blood counting plate.
The cell culture amplification method provided by the invention can be suitable for different types of tumor tissue models. For tumors with loose different structures, small surface viscosity and strong invasiveness, a considerable number of tumor cells can be obtained through physical shearing and standing settlement treatment; for a tumor with compact texture, the digestion of collagenase is matched with the further digestion of pancreatin, so that cancer cells can be further released from tumor tissues; the process flow can further improve the success rate of PDXO organoid preparation when treating some tumor tissue pieces of poor quality.
The PDXO tissue obtained by the preparation method provided by the invention is suitable for application scenes such as high-throughput drug sensitivity screening, PDXOX inoculation, in-vitro immune killing experiments, cell biology research, pathological sample preparation and research and the like. And (3) performing drug sensitivity detection screening by using the organoids, detecting the sensitivity degree of the organoids to different kinds of chemotherapeutic drugs, and guiding the administration and drug research and development.
Example 1
Unless otherwise specified, materials, reagents, and the like employed in the present invention may be obtained commercially or prepared by conventional means in the art. The raw material information used in this example is as follows:
PDXO culture medium for intestinal cancer organoid model: shanghai dredging Biotech Co., ltd; the formula comprises the following components: advanced DMEM/F12, glutaMax (1 x), B27 (1 x), N2 (1 x), penicillin-Streptomycin (1 x), 0.1mg/mL Primocin, 100ng/mL Noggin, 50ng/mL EGF, 100ng/mL Wnt3a, 500ng/mL R-spindins, 500nM A83-01, 10 μ M Y-27632, 3 μM SB202190, 1mM N-actyl-L-Cysteine, 10mM Nicotinamide, gamin, 10 ng/mLFG-2, 20ng/mL FGF-10, 1 μM PGE2;
matrigel: purchased from Corning corporation;
DMEM/F12: purchased from Gibco company;
SB202190: p38MAPK inhibitors available from MedChemExpress;
collaganase IV: purchased from Gibco company;
DNaseI: purchased from Invitrogen company;
penicillin-streptomycin (1 x): purchased from Gibco company;
primocin: purchased from InvivoGen corporation;
TrypLE: purchased from Gibco company.
The construction method of the human colon cancer organoid model PDXO comprises the following steps:
(1) Transferring the freshly extracted PDX human colon cancer tumor tissue into a biological super clean bench, removing necrotic parts and non-tumor tissue parts in the tumor tissue by using a surgical instrument subjected to aseptic treatment in a culture dish containing a fresh culture medium containing double antibodies, and shearing the necrotic parts and the non-tumor tissue parts into fragments with the diameter of about 1 mm; after being rinsed by PBS or culture medium, the tissue suspension is transferred into a sterile centrifuge tube and kept stand for 30 seconds for layering, so that tissue fragments with larger sizes are precipitated; then transferring and sucking out the supernatant containing tissue cells (without sucking out the sediment of large-size tissue fragments and keeping a small amount of liquid just covered with the tissue fragments), sieving the sucked-out supernatant cell suspension by a 70 mu m filter screen to filter the tissue or fiber fragments with larger size and only keeping single cells and cell clusters; transferring the filtrate into another centrifuge tube for preservation; then, adding a proper amount of culture medium into the residual tissue blocks precipitated at the bottom of the tube for rinsing, repeating the operations of standing, extracting the supernatant and sieving for 1 time, and enriching the filtered supernatant; centrifuging at 4deg.C and 1200rpm for 5 min, centrifuging, removing supernatant, counting cells, and preserving at 4deg.C;
(2) Dispersing the tissue sediment remained after the supernatant liquid is extracted in the step (1) in digestion liquid containing collagenase (> 250 IU/mL), transferring the tissue blocks dispersed in the digestion liquid into a culture dish or a six-hole plate, incubating for 0.5-1 hour under the condition of a shaking table of 37 ℃ and 75-100rpm, taking out the tissue every 10-15 minutes, observing the digestion condition under a microscope, and gently blowing the tissue digestion liquid by a liquid-transferring gun to help the dispersion and digestion of cell blocks in the tissue until the tissue is observed under the microscope to be mainly in a cell block consisting of a large number of single cells or tens of cells and the suspension becomes turbid without obvious large particles; when the pipette is used for blowing evenly easily, digestion is stopped by adding equal amount of PBS or culture medium; after stopping digestion, transferring the cell suspension into a new centrifuge tube, standing for 30 seconds, transferring the supernatant containing cells and cell clusters into another centrifuge tube as much as possible, and storing at 4 ℃ for standby (without sucking out large-sized tissue fragment precipitate, keeping a small amount of liquid just covered with tissue fragments);
then, 1-2mLTrypLE digestive juice is added into the tissue sediment and transferred to a cell incubator for digestion treatment at 37 ℃ for 5 minutes; after digestion is complete, dilution with >2ml pbs, and digestion is terminated;
(3) Taking out and mixing the cell suspension which is preserved at 4 ℃ for later use, combining the cell suspension with the suspension after being digested by TrypLE, sieving the cell suspension by using a cell screen with a pore size of 100 mu m, centrifuging the filtered cell suspension at 1500rpm and the temperature of 4 ℃ for 5 minutes, removing the supernatant, and re-suspending the cell mass in 20-30mL of PBS; then counting the cells, and collecting data (cell activity > 30%) related to the concentration and activity of the cells; sucking the cell suspension with the total number of living cells not exceeding 1.5X10-8 according to the number of the cells, and performing centrifugal operation; after removal of the supernatant, the centrifuge tube was cooled on ice and then resuspended in 1.2-2.0mL of matrigel (final matrigel concentration >70 wt%) to bring the concentration of viable cells in matrigel to within the range of 20,000-100,000 cells/. Mu.L; dripping matrigel drop (50 mu L/drop) containing tumor tissue cells into six hole plates at room temperature, dripping the gel drop into central region in each hole, and avoiding fusion between the gel drops, adhesion of the gel drop on hole wall or introducing air bubbles into the gel drop; after dripping the glue drop, transferring the six-hole plate back into the cell incubator, and incubating for 10-15 minutes at 37 ℃ to cause the glue drop; then adding culture medium (1.5-2.0 mL/hole) into each hole, observing under a mirror, photographing, and placing into an incubator for culturing;
(4) According to the color change of the culture medium and the growth condition of the organoids, the culture medium is replaced and supplemented every 3-4 days, and when the organoids grow full, the subculture operation is carried out; the passage method comprises the following steps: firstly removing the original culture medium, then adding 2mLPBS into each hole along the hole wall, standing for 30 seconds, and removing; then adding cell recovery liquid, 1-1.5mL of cell recovery liquid is added into each hole, and a pipetting gun is used for blowing and crushing the glue drops; transferring the pore plate onto ice or cooling in a refrigerator at 4 ℃ for 10 minutes, taking out, adding PBS (phosphate buffered saline) with equal volume or more to dilute the cell recovery liquid, further blowing the matrigel particle suspension evenly, transferring the suspension into a centrifuge tube, and centrifuging at 1500rpm and 4 ℃ for 5 minutes; removing supernatant after centrifugation, avoiding sucking away the lump part containing the organoids, using PBS to rinse the observed matrigel residue, and repeating centrifugation operation to collect organoid tissues to the greatest extent; during the passage, the organoids are dispersed in a proper amount of matrigel according to the proportion of 1:1, and glue drops are prepared, and liquid is added for culture after shaping; passaging for more than 3 times by using TrypLE digestion, and after observing normal growth of the organoid, passaging according to the concentration ratio of 500-2,000 living cells/mu L matrigel; and simultaneously, further determining parameters such as the growth speed of the organoid, removing original impurity cells in the PDX tissue, and then amplifying and freezing to obtain the human colon cancer organoid model PDXO.
FIG. 2 is a line graph of the bright field photograph and size change over time of culture of human colon cancer transplantable tumor PDXO prepared in example 1. From the figure, the human colon cancer organoid model PDXO is mainly in a three-dimensional sphere shape with a local cavity, and the size of the human colon cancer organoid model PDXO gradually increases along with the extension of the culture time, which indicates healthy growth of the human colon cancer organoid model PDXO.
Fig. 3 is a photograph of four different colon cancer PDXOs constructed in accordance with the present invention, with a scale of 200 μm. As can be seen from the figures, intestinal organoids often exhibit a saccular (internally-worn cavity) or globular three-dimensional structure.
FIG. 4 is a photograph of a bright field of a intestinal organoid constructed in accordance with the present invention (bottom left) and a photograph of a HE pathological tissue stained. From the figure, the intestinal cancer organoids show a hollow saccular tissue structure, which accords with pathological characteristics of intestinal cancer tissues.
FIG. 5 is a photograph of the bright field of a human colon carcinoma graft PDXO prepared according to the present invention (top) transferred into a 96 well plate and its bright field after 72 hours of co-incubation with Irinotecan (Irinotecan) and Fluorouracil (5-Fluorouracil) at different concentrations; wherein the upper bright field photograph has a scale of 500 μm and 200 μm (magnified), respectively, and the lower 8 Zhang Mingchang photograph has a scale of 200 μm. From the graph, the collected intestinal cancer organoids keep normal saccular structures in the 96-pore plate and are uniformly dispersed, the organoids treated by the irinotecan show obvious structural disintegration and apoptosis characteristics, and the structural disintegration and apoptosis conditions of the organoids treated by the fluorouracil are lighter under the same concentration, so that the model is more sensitive to the irinotecan.
FIG. 6 is a graph comparing the results of quantitative analyses of the sensitivity of human colon carcinoma graft PDXO shown in FIG. 5 to Irinotecan (Irinotecan) and Fluorouracil (5-Fluorouracil) using two methods, bright field photography (BF) and ATP-chemiluminescent cell viability assay kit (CellTiterGlo or CTG). Wherein the lower the IC50 value of the fit, the more sensitive the PDXO to the drug. As can be seen from the graph, according to the IC50 value results obtained by taking a photograph in the bright field and then performing statistical analysis, the irinotecan group and the fluorouracil group are respectively 0.45 μg/mL and 70.07 μg/mL, and according to the IC50 value results obtained by quantitatively detecting the CTG chemiluminescent cell viability detection kit, the irinotecan group and the fluorouracil group are respectively 2.95 μg/mL and 30.10 μg/mL, and by combining the two experimental results, it can be concluded that: the PDXM-069C human colon cancer PDXO model is more sensitive to irinotecan drugs than fluorouracil.
FIG. 7 is a graph showing the results of a test on the PDX model of human colon cancer transplantable tumor animals of the same origin as FIG. 6 for both Irinotecan (Irinotecan) and Fluorouracil (5-Fluorouracil, 5-Fu). Female BALB/c nude mice are adopted as experimental animals, and are inoculated subcutaneously on the right side of the mouse body to form tumors. Table 1 below is the statistics on day 21 for three groups of mice treated differently:
as can be seen from fig. 7 and table 1, the tumor inhibition rate of irinotecan for 21 days is 54.34% higher than that of fluorouracil in PDXM-069C human colon cancer PDX tumor model, which proves that the model is more sensitive to irinotecan, and further proves the consistency of in vitro PDXO model and in vivo PDX model in drug effect evaluation.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (10)
1. A method for constructing a patient-derived transplanted tumor organoid model PDXO, the method comprising: extracting and separating tumor tissue blocks from the PDX model, and obtaining a patient-derived transplanted tumor organoid model PDXO by using the tissue blocks through a tissue direct culture method or a cell culture amplification method;
the process of the tissue direct culture method comprises the following steps: cutting a tumor tissue block into tissue fragments and placing the tissue fragments in an organoid culture medium for culture, or embedding the tissue fragments cut by the tumor tissue block into matrigel gel drops and immersing the matrigel drops in the organoid culture medium for subculture to obtain a patient-derived transplanted tumor organoid model PDXO;
the cell culture expansion method comprises the following steps: and digesting the tumor tissue block into single cells or cell clusters with tumor dryness, re-suspending the single cells or cell clusters in a Matrigel culture medium, dripping Matrigel suspension into an orifice plate for incubation, dripping Matrigel dome glue for shaping, and culturing, changing liquid and passaging to obtain the patient-derived transplanted tumor organoid model PDXO.
2. The method of claim 1, wherein the composition of the organoid medium comprises: duplex modified Eagle Medium/nutrient mixture F-12, glutamine derivative (1 x), B27 (1 x), N2 (1 x), penicillin/streptomycin (1 x), primocin, noggin, EGF, wnt a, R-spondins, A83-01, Y-27632, SB202190, N-acetyl-L-cysteine, nicotinamide.
3. The method of claim 1 or 2, wherein the ratio of the tumor tissue mass to the organoid medium is controlled to be 0.1-1.0g:5-50mL in the tissue direct culture process.
4. A method of constructing as claimed in any one of claims 1 to 3 wherein in the process of the tissue direct culture method the culture algebra is >10 passages.
5. The method according to any one of claims 1 to 4, wherein in the cell culture expansion method, collagenase and TrypLE are used to digest the tumor tissue mass in two steps to obtain single cells or cell masses with tumor stem property.
6. The method according to any one of claims 1 to 5, wherein the two-step digestion of tumor tissue mass with collagenase, trypLE is performed by: mixing the tumor tissue block with collagenase digestive juice for digestion, stopping digestion until all cells are observed to exist in a single cell or multicellular cluster form and the mixed solution becomes turbid, centrifuging, and then adding TrypLE digestive juice into the centrifuged precipitate for digestion;
the collagenase digestive juice component comprises: collagenase IV with activity >250U/mL, DNase I with activity >250U/mL, 10mu. M Y-27632, DMEM/F12 medium, penicillin-streptomycin (1X).
7. The method of any one of claims 1-6, wherein the cell concentration of the Matrigel suspension droplets is controlled to be 20,000-100,000 viable cells/μl.
8. The method according to any one of claims 1 to 7, wherein the cell culture expansion method is performed by changing the liquid every 2 to 4 days and performing the passage every 7 to 10 days.
9. A patient-derived transplantation organoid model PDXO obtained according to the construction method of any of claims 1-8.
10. Use of a patient-derived transplanted tumor organoid model PDXO according to claim 9 in high throughput drug sensitive screening, PDXOX vaccination, in vitro immune killing experiments, pathological sample preparation.
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| CN117511880A (en) * | 2024-01-08 | 2024-02-06 | 北京大橡科技有限公司 | Method for constructing in-vitro tumor in-situ model, culture medium and in-vitro application |
| CN117625541A (en) * | 2024-01-26 | 2024-03-01 | 零壹人工智能科技研究院(南京)有限公司 | Brain glioma organoid construction method and drug sensitivity detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117511880A (en) * | 2024-01-08 | 2024-02-06 | 北京大橡科技有限公司 | Method for constructing in-vitro tumor in-situ model, culture medium and in-vitro application |
| CN117511880B (en) * | 2024-01-08 | 2024-04-19 | 北京大橡科技有限公司 | Method for constructing in-vitro tumor in-situ model, culture medium and in-vitro application |
| CN117625541A (en) * | 2024-01-26 | 2024-03-01 | 零壹人工智能科技研究院(南京)有限公司 | Brain glioma organoid construction method and drug sensitivity detection method |
| CN117625541B (en) * | 2024-01-26 | 2024-04-02 | 零壹人工智能科技研究院(南京)有限公司 | Brain glioma organoid construction method and drug sensitivity detection method |
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