CN115927164B - Culture method and application of vascularized tumor organoids - Google Patents

Culture method and application of vascularized tumor organoids Download PDF

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CN115927164B
CN115927164B CN202310075071.7A CN202310075071A CN115927164B CN 115927164 B CN115927164 B CN 115927164B CN 202310075071 A CN202310075071 A CN 202310075071A CN 115927164 B CN115927164 B CN 115927164B
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tumor
cells
culture
endothelial cells
cell
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CN115927164A (en
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刘虹余
何春花
李胜
陆政昊
王嘉
杨云旭
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Chengdu Nuoyeide Medical Laboratory Co ltd
Shenzhen Jingke Biotechnology Co ltd
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Guagnzhou Jingke Biotech Co ltd
Chengdu Nuoyeide Medical Laboratory Co ltd
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Abstract

The application belongs to the technical field of cell culture, and particularly relates to a culture method and application of vascularized tumor organoids. The method comprises the steps of taking biopsy tissue of a patient to prepare a mixed cell mass, then respectively culturing tumor cells and endothelial cells by using the mixed cell mass, finally performing 3D co-culture on the tumor cells and the endothelial cells, and inducing a vascular network in the co-cultured tumor organoid.

Description

Culture method and application of vascularized tumor organoids
The present application claims priority from chinese patent application filed 10/11/2022 [ CN2022112413917 ], entitled "method for culturing and use of vascularized tumor organoids", and is incorporated by reference in its entirety.
Technical Field
The application belongs to the technical field of cell culture, and particularly relates to a culture method and application of vascularized tumor organoids.
Background
It is found that a great amount of new blood vessels are formed in the tumor during the occurrence and development process, and the new blood vessels provide nutrition and moisture for the growth of the tumor, and simultaneously create conditions for diffusing tumor cells to a far place and forming new metastasis at different parts in the body. Taking breast cancer as an example, the high proliferation and metastasis properties of breast cancer depend on the newly formed blood vessels, so inhibiting angiogenesis is a key research content for overcoming the invasiveness of the breast cancer tumor microenvironment. The existing research consensus kick on has established the concept of a tumor-angiogenesis model, but most of the current researches adopt umbilical vein endothelial cells (HUVEC), mesenchymal cells (MSC) and the like as vascular endothelial sources, and the umbilical vein endothelial cells, the mesenchymal cells (MSC) and the like are added into a breast cancer cell line to carry out animal in vitro transplantation tumor models so as to achieve the purpose of angiogenesis. The model established by the method depends on animals, namely, the model needs to be transplanted on the animals, so that the modeling period is long and the cost is high. Secondly, as HUVEC and MSC cell lines have undergone long-term culture and passage, their molecular and phenotypic characteristics have changed, and thus co-culture with HUVEC and MSC cell lines and breast cancer cell lines often faces microenvironments where they cannot completely mimic interactions between tumors and blood vessels in vivo due to lack of vascular properties in tumor microenvironments, nor have tumor heterogeneity between different patients. Thus, animal models of "tumor-angiogenesis" constructed based on HUVECs and MSCs often fail to convert completely efficiently to clinic.
The recently developed tumor organoid (PDO) model is widely used in preclinical tumor research and clinical drug screening due to its feature of maintaining tumor heterogeneity and high modeling success rate. Current research suggests that tumor organoids have potential as an in vitro assay platform for predicting response to personalized immunotherapy. However, due to the lack of important physiological processes in vivo and the lack of microenvironments such as immune cells and mesenchymal cells, the organoids constructed in vitro cannot fully replicate all cell types and maturity of real organs, nor are there distributions of blood vessels, lymphatic vessels and nervous systems, thus making it difficult for organoid models to evaluate the true therapeutic response and pharmacological mechanisms of immunomodulators.
At present, although technical schemes for carrying out co-culture on endothelial cells and organoids to induce organoid angiogenesis are attempted, the technical schemes have complex operation and long culture time, and meanwhile, the problem of maintaining heterogeneous inheritance of original tumor tissues of different patients cannot be solved. For example, patent application number CN2018800224776, entitled "stable three-dimensional angiogenesis and methods of forming same", discloses a technique for vascularizing organoids or decellularized organs by culturing the organoids or decellularized organs with endothelial cells comprising an exogenous nucleic acid encoding an ETV2 transcription factor under conditions that allow expression of the exogenous ETV2 protein in the endothelial cells. The protocol first requires at least 3-4 weeks to construct an endothelial cell that encodes an exogenous nucleic acid encoding an ETV2 transcription factor (i.e., to construct a tailored endothelial cell), and the endothelial cell source in the method includes HUVEC cell lines (lacking tumor microenvironment vascular properties). After the construction of the endothelial cells, the endothelial cells are further co-cultured with the organoids, and the endothelial cells need to express ETV2 protein under culture conditions to induce the formation of a luminal, stable three-dimensional blood vessel. Therefore, the technical scheme for constructing stable three-dimensional angiogenesis provided by the method is complex to operate and takes a long time. More importantly, the patent method is to surgically implant the vascularized organoid constructed in vitro into the patient, rather than constructing a platform for preclinical drug screening in vitro.
In view of the foregoing, there is a need to develop a method and model that can access the angiogenic properties of tumors in different patients and provide effective support for preclinical screening of anti-angiogenic drugs in an effort to alleviate at least one of the problems of the prior art.
Disclosure of Invention
In view of the above, the present application aims to provide a culture method and application for inducing vascularization of tumor organoids, and the specific technical scheme is as follows.
A culture method for inducing vascularization of a tumor organoid, comprising the steps of:
step one: taking biopsy tissue of a patient, cleaning, shearing, then performing tissue digestion, and stopping the tissue digestion after 0.5-2 hours to obtain a mixed cell mass (the cell mass at the moment comprises tumor cells, a small amount of endothelial cells, immune cells, stromal cells and the like, so-called mixed cell mass);
step two: preparing the mixed cell mass obtained in the step one into a cell suspension, adding matrigel to resuspend and precipitate, inoculating the cell suspension into a pore plate, adding a tumor cell culture medium to amplify and culture tumor cells, and carrying out passage after the density of the tumor cells is fused by at least 80 percent (the total number of tumor organoids and the total number of cells fused with each other are calculated under the same visual field to obtain a fusion ratio, and counting a plurality of visual fields to obtain an average value of the cell density fusion ratio);
step three: preparing the mixed cell mass obtained in the first step into a cell suspension, adding an endothelial cell culture medium to re-suspend and precipitate, inoculating the cell suspension into a culture plate coated with gelatin, culturing to obtain endothelial cells, and carrying out passage after the endothelial cells are fused at least 80% (calculating the cell density fusion percentage and the second step);
step four: performing magnetic screening by using immunomagnetic beads or purifying the endothelial cells by using a flow cytometer until the purity of the endothelial cells reaches more than 90% (the purity of the endothelial cells can influence the accuracy of experimental results;
step five: mixing and re-suspending the tumor cells obtained in the second step and the endothelial cells obtained in the fourth step in matrigel, wherein the proportion of the re-suspended mixed cells in matrigel is 4000-10000/25 μl, and further inoculating the matrigel into a co-culture medium for 3D co-culture, wherein the co-culture medium consists of FBS, green/streptomycin, L-Glutamate, DMEM/F12 basal medium and angiogenesis promoting components, and the angiogenesis promoting components comprise VEGF and vitamin C (two processes are included in the co-culture process in this case, namely, tumor organoids are formed and vascular networks are formed inside the tumor organoids and between organoids after being induced by endothelial cells);
step six: immunofluorescent staining identification is carried out on the blood vessels generated in the co-culture system.
It can be understood that the composition of the tumor cell culture medium adopted in the method is different according to different tumor tissue sources, and the composition of the culture medium is configured according to the prior literature so as to achieve the purpose of culturing tumor cells to form tumor organoids.
The "co-culture" in the method of the present application is 3D co-culture of tumor cells with endothelial cells. If only two kinds of cells are mixed and then subjected to conventional 2D co-culture, there is a high possibility that tumor cells and endothelial cells will grow next to each other, and blood vessels cannot be effectively differentiated. However, after forming a 3D structure, tumor cells form a tumor organoid, and endothelial cells induce angiogenesis of the tumor organoid, a vascular network may be formed between tumor organoid clusters or between clusters, so that part of microenvironment of the tumor organoid is simulated, a model which is closer to the actual behavior pattern of the tumor is constructed on an in vitro platform, and higher accuracy is provided for subsequent preclinical drug screening.
Further, in the fifth step, the mixing ratio of the tumor cells to the endothelial cells is 1:1 to 1:2.
preferably, the mixing ratio of the tumor cells and the endothelial cells comprises 1:1 or 1:2.
Further, in the first step, the biopsy tissue of the patient is washed with a washing liquid comprising 1% blue/streptomycin and 99% DPBS buffer by volume.
Further, in the first step, tissue digestion is performed by using a tissue digestion solution, wherein the tissue digestion solution comprises 2% of cyan/streptomycin, 97% of DPBS buffer, 1% of BSA by mass fraction and 1mg/ml of type I collagenase by volume ratio.
Further, in the first step, tissue digestion is stopped with a stop solution comprising 10% FBS and 90% DMEM/F12 medium.
Further, the co-culture medium in the fifth step consists of 10% by volume of FBS, 1% by volume of green/streptomycin, 0.2mM L-Glutamate, 89% by volume of DMEM/F12 basal medium, 100ng/ml VEGF and 60. Mu.g/. Mu.l vitamin C.
Preferably, L-Glutamate is replaced with Glutamax because L-Glutamate is unstable in the medium.
The co-culture medium provided by the method not only needs to provide nutrients and cytokines required for maintaining the growth of tumor cells and endothelial cells, but also provides components capable of inducing tumor organoids to generate blood vessels.
Further, the co-cultured mixed cells in the fifth step are cultured in 5% CO 2 、21％O 2 Is subjected to normoxic conditions and 5% CO 2 、2％ O 2 Is alternately cultured under low oxygen conditions.
The vascularized tumor organoid model obtained by the culture method is characterized in that the vascularized tumor organoid model is a co-culture model of endothelial cells from the same patient for inducing tumor organoids to generate blood vessels.
The vascularized tumor organoid model obtained by the method forms an interactive network of tumor and blood vessel related cells, but not a blood vessel tissue which can completely reduce and transport nutrient substances and oxygen.
Further, the source of tumor cells of the tumor organoid includes breast cancer, lung cancer, liver cancer, bile duct cancer, stomach cancer, intestinal cancer, bladder cancer, kidney cancer, pancreatic cancer or esophageal cancer.
Further, the vascularized tumor organoid model is applied to preclinical screening of anti-angiogenic drugs.
Further, the vascularized tumor organoid model is applied to the preparation of an in vitro anti-tumor angiogenesis drug screening platform.
Preferably, the anti-angiogenic drugs include, but are not limited to, hsp90 inhibitors and/or multiple kinase inhibitors of Raf-1 and B-Raf.
Beneficial technical effects
The existing research finds that the sensitivity of endothelial cells to drugs is more truly reflected, and can not be realized by simply using a single cell line, so that the accuracy and precision of the existing many preclinical drug screening models are not enough. In order to simulate a microenvironment close to the interaction between a tumor and a blood vessel in a human body and also remove heterogeneity among individuals, the application further provides a detection platform with higher in-vitro drug screening precision, which is suitable for personalized accurate medical treatment. In the vascularized tumor organoid model provided by the application, original tumor cells and endothelial cells are derived from the same patient. Since endothelial cells within a tumor are different from those in normal tissues, they also exhibit different characteristics depending on the malignancy of the tumor, and thus have inter-individual and intra-individual heterogeneity. Through long-term exploration, the tumor cells and endothelial cells from the same patient are used for 3D co-culture, and the constructed model of the individual tumor organoid-endothelial cells can truly restore vascular networks and microenvironments in tumor tissues in vitro in a short time (10 days) after induction, can well restore heterogeneity among individuals and in individuals, further remarkably improves the accuracy and precision of a preclinical drug screening detection platform, and has great potential in promoting the problem of individual accurate medical development.
In the research and development process, the team of the application finds that the co-culture of the tumor cells and the endothelial cells of the same patient has a certain technical difficulty, including the great difficulty of the primary tumor cells, and the sorting and purification and culture of the endothelial cells of the tumor sources also have a certain difficulty. For example, the purity of endothelial cells can affect the final vascularized network formed; for another example, the culturing of tumor cells into tumor organoids requires 3D culture, while the characteristics of adherent growth of endothelial cells make it necessary to perform 2D culture, so that co-culturing tumor cells and endothelial cells requires simultaneous satisfaction of the different growth characteristics of both cells. Through a large number of growths and experiments, the team of the application cultures tumor organoids and 2D cultured endothelial cells respectively, then further sorts and purifies the endothelial cells, finally carries out 3D co-culture on the two cells, and realizes that the tumor organoids with vascular network differentiation are cultured in a short time (10 days) by optimally setting a co-culture medium with specific tumor cell-endothelial cell proportion, specific components, specific culture conditions and the like, and the heterogeneity among individuals and inside the individuals can be reduced well.
In general, the culture method provided by the application is convenient to operate, short in modeling time and high in modeling success rate (taking the blood vessel network differentiated from the tumor organoid as a standard for modeling success), and the problems that other technical platforms need expensive instruments and complicated operation steps and have high technical thresholds are avoided without additionally resorting to genetic engineering technology and micro-fluidic platforms. Can be better and more widely applied to basic scientific research.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the application and that other drawings may be derived from these drawings without inventive faculty.
FIG. 1 is a schematic diagram of the overall technical route of the cultivation method of the present application;
FIG. 2 is a chart of vascularized fluorescent staining of different tumor organoids (breast, bladder and liver cancer in sequence from left to right; wherein endothelial cells are stained with CD31 antibody, which is indicated as green fluorescence; tumor cells are stained with Pan-CK antibody, which is indicated as red fluorescence; DAPI stains the nucleus, which is indicated as blue fluorescence, indicating that all cells, non-cellular components, have no blue fluorescence);
FIG. 3 is a bright field plot of tumor cells and endothelial cells of the same patient source at a passaging density > 80% (scale bar: 100 μm);
FIG. 4 is a graph of the open field of cells at different plating ratios (from left to right: cell number < 4000, cell number 4000-10000, cell number > 10000; scale: 100 μm);
FIG. 5 is a graph showing the effect of 3D co-cultivation using co-cultivation medium without and with added pro-angiogenic components, respectively (scale: 50 μm);
FIG. 6 is a graph showing experiments with 17-AAG treatment after 3D matrigel co-culture of tumor organoids and endothelial cells, and count plating;
FIG. 7 is a graph showing experiments with Sorafenib treatment after 3D matrigel co-culture of tumor organoids and endothelial cells, counting plating;
FIG. 8 is a graph showing the difference in endothelial network formation after the action of 17-AAG at high, medium and low drug concentrations (scale: 50 μm);
FIG. 9 is a graph showing the difference in endothelial network formation after Sorafenib has been administered at high, medium and low drug concentrations (scale: 50 μm).
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. It will be apparent that the described embodiments are some, but not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.
In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, a rangeThe description of (c) should be taken as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.
The vascularized tumor organoids described herein induce tumor organoids to form blood vessels on an in vitro constructed organoid platform.
The organoid refers to an in-vitro model with tissue morphology formed by cell self-assembly, and has a certain function because the generation of partial vascular network is induced.
The accuracy of the application refers to that the vascularized tumor organoid cultivated by the method has certain vascular network differentiation, and is closer to the real intratumoral environment, so that the vascularized tumor organoid has higher accuracy in response to drug sensitivity and efficacy compared with the common organoid model when being used for preclinical drug screening.
The "accuracy" of the present application means that the vascularized tumor organoids cultured by the method of the present application have accuracy that can reflect drug sensitivity and efficacy of different patients compared to a general drug screening model when used in preclinical drug screening due to the inter-and intra-individual heterogeneity.
The Matrigel is Matrigel.
DMEM/F12, fetal Bovine Serum (FBS), trypLE, type I collagenase, DPBS, L-Glutamate, blue/streptomycin from ThermoFisher, endothelial cell culture medium ECGM-MV from Promocell, VEGF from PreproTech, gelatin from the holothurian biotechnology, matrigel from Corning, human CD31 immunomagnetic beads from Miltenyi Biotec, cleaning solution, tissue digestion solution, and stop solution are products developed by Chenopodial medicine.
Example 1
1. Preparation of the Experimental Material
DMEM/F12, fetal Bovine Serum (FBS), trypLE, collagenase type I, DPBS, HPBS, BSA, L-Glutamate, green/streptomycin, trypsin-EDTA was purchased from thermosipher.
Endothelial cell culture medium ECGM-MV was purchased from Promocell and its components included final concentrations of 0.05ml/ml FBS,0.004ml/ml ECGS,10ng/ml EGF, 90. Mu.g/ml heparin and 1. Mu.g/ml hydrocortisone.
Tumor cell culture media are described in various literature and are suitable for use with specific tumor types.
Matrigel was purchased from corning.
VEGF was purchased from PreproTech.
Gelatin was purchased from the next holy biotechnology.
Human CD31 immunomagnetic beads were purchased from Miltenyi Biotec.
2. Preparation of Medium and other reagents
1) Cleaning liquid: comprises a volume ratio of 1% green/streptomycin and 99% DPBS buffer.
2) Tissue digestion solution: comprises 2% of blue/streptomycin, 97% of DPBS buffer solution, 1% of BSA by mass fraction and 1mg/ml of type I collagenase by volume ratio.
3) Stop solution: comprises 10% FBS and 90% DMEM/F12 medium.
4) Co-culture medium:
example 2
An example of a method of inducing tumor organogenesis
1) Sample source: the biopsy tissue source was ethical and the patient was aware and agreed that fresh samples obtained on the day of the patient's surgery were stored temporarily in a preservative fluid (4 ℃, <24 h). The tumor sample sources in this embodiment include breast cancer tissue, bladder cancer tissue and liver cancer tissue.
Tissue pretreatment: the tissue was washed 3 times with washing liquid, adipose tissue was removed and tissue was prevented from collecting necrotic and hypoxic areas, and the tissue was cut into two parts with sterile surgical scissors, one part (tissue a) for separating cultured tumor cells and the other part (group B) for separating cultured endothelial cells.
2) Tissue digestion: the tissues A and B were minced, and incubated with 8-10ml of tissue digests, respectively, in two 50ml centrifuge tubes A, B,37℃water bath for 0.5-2h.
Collecting cell mass: removing excessive tissue fragments by using a 100 μm cell filtration mesh screen, cleaning the mesh screen once by using a cleaning solution, adding 2ml of stop solution to stop digestion, transferring the cell suspension to a 15ml centrifuge tube, centrifuging 300g for 5min, and discarding the supernatant; tube B was terminated by adding 2ml of stop solution without filtration, transferring to a 15ml centrifuge tube, and centrifuging the supernatant for 5 min. If the bottom of the tube has red blood cells aggregated, then 1ml of red blood cell lysate is added to lyse red blood cells (2 min on ice), then 4ml of cleaning solution is added to stop, 300g is centrifuged for 5min, and the supernatant is discarded.
3) Tumor cell culture and expansion (3D): the matrigel was added to the A tube to re-suspend the pellet, the cell-containing gel droplets were inoculated into a 48-well plate (25. Mu.l/well), and tumor cell medium was added for culture. The cells were observed under a microscope to fuse to at least 80% of density for passaging. See fig. 3.
4) Endothelial cell culture (2D): and (3) adding an endothelial cell culture medium into the B tube to re-suspend and precipitate, and inoculating the cell suspension into a culture plate coated with gelatin for 2D culture. Cells to be fused to at least 80% were observed under a microscope and passaged. See fig. 3.
The endothelial cells have the characteristic of adherent growth, and are isolated and cultured from the biopsy tissue, so that more endothelial cells are isolated from tumor tissues by utilizing conditions such as an endothelial cell culture medium and gelatin coating, and meanwhile, non-target cells cannot be grown in an adherent manner and are suspended in the culture medium, the endothelial cells can be eluted better, and the purity of the endothelial cells cultured later is guaranteed to be kept at a higher level.
5) Endothelial cell screening and expansion: after 0.25% trypsin/EDTA digestion (< 1 min), cells were collected and stopped using stop solution, supernatant was discarded by 5min centrifugation, 100 μl of PBS containing 2% FBS was added to resuspend pellet, and purification was performed by immunomagnetic bead screening or flow cytometry. Wherein the immunomagnetic beads can be CD31 labeled immunomagnetic beads, and when the purity of endothelial cells reaches 90%, the immunomagnetic beads are inoculated into a gelatin coated pore plate, and endothelial cell culture medium is added for maintenance culture.
6) 3D co-culture: amplifying and culturing the obtained tumor cells and endothelial cells, respectively digesting with TrypLE and pancreatin, respectively adding 2 times volume of cleaning solution and equal volume of stop solution to stop digestion, centrifuging, discarding supernatant, respectively adding 2ml of FPBS to resuspend precipitate, counting, mixing tumor cells and endothelial cells with determined cell number according to the required inoculation amount, making into two cell suspensions, centrifuging, resuspending with matrigel, inoculating in 48-well plate, adding3D co-culturing in co-culture medium at 37deg.C and 5% CO 2 ，21％O 2 After 24h, the cell culture plates were transferred to 37℃with 5% CO 2 ，2％O 2 Hypoxia culture was performed in a hypoxia incubator to induce uniform and rapid vessel formation, and after 3 days, the cell culture plates were returned to 37℃with 5% CO 2 、21％O 2 The culture was continued for 10 days.
7) Immunofluorescent staining was performed on the vessels generated in the co-culture system (vascularization evaluation).
The washing liquid of the culture method comprises a DPBS buffer solution with the volume ratio of 1% of cyan/streptomycin and 99%.
The tissue digestion solution of the culture method comprises 2% of cyan/streptomycin, 97% of DPBS buffer solution, 1% of BSA by mass fraction and 1mg/ml of type I collagenase by volume ratio.
The stop solution of the culture method comprises 10% FBS and 90% DMEM/F12 medium.
Endothelial cell culture plates of the above culture methods were 6-well plates, coated with 1% or 1.5% gelatin in 1 XPBS for 1 h.
The endothelial cells were cultured and expanded in 2D using endothelial cell culture Medium (Medium comprising 0.05ml/ml FBS,0.004ml/ml ECGS,10ng/ml EGF, 90. Mu.g/ml heparin, 1. Mu.g/ml hydrocortisone) at a final concentration of Growth Medium MV.
The ratio of endothelial cells to tumor cells in the co-cultured mixed cells was 1:1, and resuspended in matrigel at a concentration of 8-10 mg/ml (about 4000-10000/25. Mu.l).
The co-culture medium contained 10% FBS by volume, 1% Green/Streptomycin by volume, 0.2mM L-Glutamate by volume, 100ng/ml VEGF, 60. Mu.g/. Mu.l vitamin C and 89% DMEM/F12 basal medium by volume, wherein VEGF and vitamin C were pro-angiogenic components.
Matrigel used in the above culture method is Matrigel.
FIG. 1 shows a schematic of the technical route of the present application, wherein anti-vascularization detection means that the generated vascularized tumor organoid model is further used for screening and detection of preclinical anti-vascularization drugs.
Example 3
An example of a method for inducing breast cancer tumor organogenesis angiogenesis is provided, comprising two parts, i, ii.
And I, separating and obtaining endothelial cells and breast cancer tumor cells from tissue biopsy sources of patients.
Before separating endothelial cells and tumor cells, a cleaning solution, a tissue digestion solution and a digestion stop solution (i.e. a stop solution) are prepared first.
Preparation of gelatin coated 6-well plates: gelatin was prepared as a 2% gelatin solution in a conical flask according to the manufacturer's method of use, placed in an autoclave, autoclaved at 121 ℃ for 20min, and at 15psi. The 6-well plate was coated after dilution with 1 XPBS to a 1.5% gelatin solution.
Tissue biopsy samples were obtained with ethical requirements and patient awareness and consent. Samples were stored in 15ml centrifuge tubes with preservation solution at 4 ℃ for less than 24 hours. Taking out the tissue from the preservation solution, uniformly mixing the preservation solution, taking 100 mu l of the mixture, observing under a mirror, judging the cell shedding condition and the cell activity, transferring a tissue sample into a 50ml centrifuge tube, adding 10ml of cleaning liquid, vibrating the centrifuge tube to clean the tissue (repeated 3 times), using a sterile surgical scissors and forceps to divide the tissue into two parts (A/B), using the tissue A to separate tumor cells, using the tissue B to separate endothelial cells, putting the tissue blocks into two culture dishes, shearing, adding 8-10ml of tissue digestion solution, digesting for 1h in a 37 ℃ water bath, adding 2ml of termination solution to terminate the digestion, centrifuging for 300g and 5min, discarding the supernatant, cleaning the sediment for 2 times by using FPBS, finally embedding the A cell sediment into Matrigel (4 or more than 4 are 1 cell clusters, 1000 cell clusters/25 mu l), and inoculating the sediment into a 48-well plate. After resuspension of the B cell pellet with endothelial cell culture medium, it was seeded in gelatin-coated 6-well plates.
Purification of endothelial cells was performed 24-72h later, and the present example uses CD31 beads for screening, comprising the following steps: the cells were collected in 15ml centrifuge tubes, centrifuged for 3min at 300g, and the cell pellet was resuspended in 1-2ml medium for cell counting.300g, centrifuging for 3min, discarding supernatant, 1×10 7 cells were resuspended in 60. Mu.l medium and screened according to the manufacturer's method. The endothelial cells after screening are inoculated in a 6-well plate coated with gelatin and cultured and expanded to about 10 with endothelial cell culture medium 5 Subsequent experiments can be performed at various times.
Culturing and expanding tumor cells to 2-3 generations, and expanding cell number to about 10 5 About each cell, endothelial cells and tumor cells were co-cultured in suspension.
Preferably, the preservation solution and tissue washing solution are collected, centrifuged for 5min at 300g, the supernatant is discarded, and the pellet and digested cell pellet are seeded together in a 48-well plate to maximize the collection of tumor cells.
Preferably, the digestion time is controlled between 0.5 and 2 hours, and the state and quantity of dissociated cells are observed by pipetting with a 10ml pipette at intervals of 10 minutes.
Preferably, the DPBS can be replaced by DHanks, which protects the cell viability index more strongly.
Preferably, endothelial cells are purified by a variety of screening methods, such as flow cytometry.
Co-culturing endothelial cells and tumor cells.
It is desirable to prepare in advance a co-culture medium consisting of FBS, green/streptomycin, L-Glutamate, DMEM/F12 basal medium and an angiogenesis promoting component including VEGF and vitamin C.
Preferably, L-Glutamate is replaced with Glutamax because L-Glutamate is unstable in the medium.
Tumor cells and endothelial cells were collected separately, resuspended and pelleted with co-culture medium, counted, the two cells were mixed in a 1:1 ratio (48 well plate, about 4000 cells/well), 300g, centrifuged for 5min, the supernatant discarded, resuspended with appropriate amount of Matrigel (48 well plate, 25 μl/well), after gel drop coagulation, 300 μl of pre-formulated co-culture medium per well was added, at 37 ℃,5% co 2 ，21％O 2 After 24h, the cell culture plates were transferred to 37℃with 5% CO 2 ，2％O 2 Hypoxia culture was performed in a hypoxia incubator to induce uniform and rapid vessel formation, and after 3 days, the cell culture plates were returned to 37℃with 5% CO 2 、21％O 2 The culture was continued for 10 days.
The method can always perform co-culture of endothelial cells and tumor cells in a normoxic state, and finally can detect endothelial networks, but the endothelial networks after hypoxia treatment are more and the distribution in aggregates is more uniform. Network formation of the endothelium will be detected on day 4 and the aggregation of endothelial cells with tumor cells will be detected on day 7, since the seeding of the gel drop, at which time the endothelial network expands in bulk in the tumor cell aggregates.
Endothelial network detection:
the organoid-containing glue droplets were immunofluorescent stained by co-cultivation to about day 10, and stained with CD-31, pan-CK antibodies, and DAPI staining solution according to conventional staining procedures, and photographed under a fluorescent microscope, as shown in FIG. 2. It can be seen that endothelial cells and tumor cells co-occur in matrigel and cross each other, and that endothelial cells form a vascular-like structure both inside and outside the organoid. This 3D co-culture format was shown to be successful and to some extent mimics the interaction of human tumors with the vascular network.
Example 4
The vascularized tumor organoid model drug screening experiment constructed by the application
Anti-angiogenic drugs, such as 17-AAG and Sorafenib, were added to the constructed model and immunofluorescent staining was used to detect changes in endothelial network.
Wherein, 17-AAG is also called Tanespimycin, CP 127374, is a potent Hsp90 inhibitor, and in addition, 17-AAG can inhibit Akt activation and HER2 and ErbB2 expression, and has obvious anti-tumor activity in vitro.
Sorafenib (BAY 43-9006, NSC-724772) is a multiple kinase inhibitor of Raf-1 and B-Raf. Sorafenib also inhibits VEGFR-2, VEGFR-3, PDGFR-beta, flt-3 and c-KIT. Sorafenib induces autophagy, apoptosis and activates ferroptosis, and has antitumor activity in vivo.
In the embodiment, after the tumor organoids and endothelial cells are subjected to 3D matrigel co-culture, counting and plating are respectively carried out on 17-AAG and Sorafenib (Sorafenib) drug treatment, 7 drug gradients are set, the sensitivity degree and IC50 data of the tumor organoids to the two drugs are obtained, and a certain basic data support is provided for subsequent clinical medication and drug dosage.
The data for the detection of the added drug in this embodiment are shown in the following table:
17-AAG
concentration (nM) 300 150 75 37.5 18.75 9.375 4.6875
Survival rate 3.41 4.98 20.87 91.57 100.75 103.98 103.80
Sorafenib
Concentration (nM) 400 200 100 50 25 12.25 6.125
Survival rate 2.14 6.00 31.50 75.78 87.92 96.36 102.60
Conclusion of experiment: as shown in the table above, the survival rate of tumor cells increases with decreasing concentration of drug added, with a dose-dependent effect. The model can be used for screening preclinical relevant medicines, and experimental diagrams of relevant results are shown in fig. 6 and 7.
Fig. 8 and 9 show the changes in endothelial network after addition of high, medium and low 17-AAG or Sorafenib, respectively, and it can be seen from the figures that green fluorescence representing angiogenesis decreases after addition of high concentration of drug (left panel), indicating that angiogenesis increases with decreasing drug concentration. Thus, it was consistent with the results shown in the pharmaceutical experimental table.
Example 5
Co-culture medium composition effect verification
The co-culture medium provided by the application consists of 10% FBS (FBS) in volume ratio, 1% green/streptomycin in volume ratio, 0.2mM L-Glutamate, 89% DMEM/F12 basal medium in volume ratio, 100ng/ml VEGF and 60 mug/mul vitamin C, wherein VEGF and vitamin C are pro-angiogenesis components. This example demonstrates the effect of adding and removing the pro-angiogenic component on co-culture results, with the other formulation unchanged. See fig. 5.
Experimental results: in the figure, green represents the blood vessels produced, red represents tumor cells, blue represents nuclei, indicating that all cells, non-cellular components, are devoid of blue fluorescence. Obviously, only tumor cells and small amounts of endothelial cells were seen without the addition of the pro-angiogenic component (left panel) during the same incubation time, whereas a large number of vascular network structures were clearly seen after the addition of the pro-angiogenic component (right panel).
Example 6
Verification of different plating ratios
In the co-culture method provided by the application, the proportion of the resuspended mixed cells in the matrigel is 4000-10000/25 μl. This example demonstrates the structure of different plating ratios, see fig. 4.
Experimental results: as can be seen, when the number of co-cultured cells is less than 4000/25. Mu.l, the density of tumor cells and endothelial cells is low, and the tumor and endothelial mixed organoids are difficult to interact; when the number of the co-cultured cells is 4000-10000/25 mu l, the tumor cells and the endothelial cells are fused and grown, so that the tumor organoids with vascular networks are effectively formed; when the number of co-cultured cells is more than 10000 cells/25. Mu.l, the cell density is too high to form competitive inhibition, and the cells cannot effectively proliferate, failing to form a mixed tumor and endothelial organoid. It can be seen that the cell density of the plating has a great influence on the growth of cells, the density is too low, the cells are difficult to survive and proliferate, the density is too high, the cells compete with each other for nutrients, contact inhibition is generated, and the cells are difficult to proliferate effectively. 4000-10000/25 ul is the optimal number of cells to ensure efficient proliferation of the co-cultivated organoids, more or less than this is more difficult to achieve.
The embodiments of the present application have been described above with reference to the accompanying drawings, but the present application is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present application and the scope of the claims, which are to be protected by the present application.

Claims (6)

1. The culture method of the vascularized tumor organoid model is characterized in that the vascularized tumor organoid model is a co-culture model of endothelial cells from the same patient for inducing tumor organoid to generate blood vessels, and comprises the following steps:
step one: taking biopsy tissue of a patient, cleaning, shearing, then performing tissue digestion, and stopping the tissue digestion after 0.5-2 hours to obtain a mixed cell mass;
step two: preparing the mixed cell mass obtained in the step one into cell suspension, adding matrigel to re-suspend and precipitate, inoculating into a pore plate, adding a tumor cell culture medium to amplify and culture tumor cells, and carrying out passage after the density of the tumor cells is fused by at least 80%;
step three: preparing the mixed cell mass obtained in the step one into cell suspension, adding an endothelial cell culture medium to re-suspend and precipitate, inoculating the cell suspension into a culture plate coated with gelatin, culturing to obtain endothelial cells, and carrying out passage after the endothelial cell density is fused by at least 80%;
step four: further amplifying and culturing the endothelial cells obtained in the step three, and magnetically screening the endothelial cells by using immunomagnetic beads or purifying the endothelial cells by using a flow cytometer until the purity of the endothelial cells reaches more than 90%;
step five: mixing and re-suspending the tumor cells obtained in the second step and the endothelial cells obtained in the fourth step in matrigel, wherein the ratio of the re-suspended mixed cells in matrigel is 4000-10000/25 μl, and the mixing ratio of the tumor cells and the endothelial cells is 1: 1-1: 2, further inoculating into a co-culture medium for 3D co-culture, wherein the co-culture medium consists of FBS, green/streptomycin, L-Glutamate, DMEM/F12 basal medium and angiogenesis promoting components, and the angiogenesis promoting components comprise VEGF and vitamin C; the mixed cells were at 5% CO 2 、21%O 2 Is subjected to normoxic conditions and 5% CO 2 、2%O 2 Alternatively culturing under low oxygen condition;
step six: immunofluorescent staining identification is carried out on the blood vessels generated in the co-culture system.
2. The method of claim 1, wherein in step one, the patient biopsy is washed with a wash solution comprising 1% green/streptomycin and 99% DPBS buffer by volume.
3. The method according to claim 1, wherein in the first step, tissue digestion is performed with a tissue digestion solution comprising 2% by volume of blue/streptomycin, 97% by volume of DPBS buffer, 1% by mass of BSA and 1mg/ml of collagenase type I.
4. The culture method of claim 1, wherein in the first step, tissue digestion is terminated with a termination solution comprising 10% FBS and 90% DMEM/F12 medium.
5. The culture method according to claim 1, wherein the co-culture medium in the fifth step consists of 10% by volume of FBS, 1% by volume of green/streptomycin, 0.2mM L-glutarate, 89% by volume of DMEM/F12 basal medium, 100ng/ml VEGF and 60. Mu.g/μl vitamin C.
6. The method of claim 1, wherein the source of tumor cells of the tumor organoid comprises breast cancer, lung cancer, liver cancer, cholangiocarcinoma, stomach cancer, intestinal cancer, bladder cancer, kidney cancer, pancreatic cancer, or esophageal cancer.
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