阅读说明：本技术 一种基于生物三维打印的体外gbm侵袭模型的构建方法 (Method for constructing in-vitro GBM (GBM) invasion model based on biological three-dimensional printing ) 是由 马梁 李雨亭 吴钰桐 张斌 杨华勇 于 2019-08-02 设计创作，主要内容包括：本发明公开了一种基于生物三维打印的体外胶质母细胞瘤(GBM)侵袭模型的构建方法,属于组织工程和生物三维(3D)打印领域。此方法采用含细胞的水凝胶作为“生物墨水”,利用生物三维打印机构建体外GBM侵袭模型。模型结构由载有正常人脑胶质细胞的水凝胶打印而成,并接种神经胶质瘤细胞团。此模型对研究GBM的侵润性生长具有重要意义,有望为多形性神经胶质母细胞瘤的治疗方法研究提供帮助。(The invention discloses a method for constructing an in-vitro glioblastoma multiforme (GBM) invasion model based on biological three-dimensional printing, and belongs to the field of tissue engineering and biological three-dimensional (3D) printing. The method adopts hydrogel containing cells as biological ink and utilizes a biological three-dimensional printer to construct an in-vitro GBM invasion model. The model structure is printed by hydrogel loaded with normal human brain glial cells, and is inoculated with glioma cell mass. The model has important significance for researching the invasive growth of GBM, and is expected to provide help for the research of the treatment method of the glioblastoma multiforme.)

1. A method for constructing an in-vitro GBM invasion model based on biological three-dimensional printing is characterized by comprising the following steps:

1) Preparing biological ink containing human brain glial cells; the solvent of the biological ink is purified water, and the components of the biological ink comprise hyaluronic acid, alginate and gelatin; the content of the hyaluronic acid in the bio-ink is 0.01-0.02g/ml, the content of the alginate in the bio-ink is 0.01-0.02g/ml, and the content of the gelatin in the bio-ink is 0.05-0.15 g/ml;

2) loading the biological ink into a feeding system of a multi-nozzle micro-extrusion biological three-dimensional printer, and printing a support structure according to a specified path; impregnating the obtained support structure with a calcium chloride solution;

3) Uniformly inoculating GBM cell masses into the scaffold structure obtained in the step 2);

4) Placing the support structure inoculated with the GBM into a cell culture box for continuous culture, changing the liquid every 2-3 days to obtain an in-vitro GBM invasion model with high cell survival rate, and respectively extracting RNA of the culture structure for sequencing on 1 st, 7 th and 14 th days of culture.

2. The method for constructing the in vitro GBM invasion model based on biological three-dimensional printing according to claim 1, wherein the selected content of hyaluronic acid in the bio-ink is 0.02g/ml, the selected content of alginate in the bio-ink is 0.02g/ml, and the selected content of gelatin in the bio-ink is 0.010 g/ml.

3. the method for constructing the in vitro GBM invasion model based on biological three-dimensional printing according to claim 1, wherein the biological ink further comprises glucose as an invasion inducing factor, and the addition amount of the glucose is 0.005-0.02 g/ml.

4. the method for constructing the in-vitro GBM invasion model based on biological three-dimensional printing according to claim 1, wherein the scaffold structure is a single-layer, multi-layer grid structure or a three-dimensional framework structure.

5. the method for constructing the in vitro GBM invasion model based on biological three-dimensional printing according to claim 1, wherein in the step 4), the culture medium used in the culture process is prepared from a common cell culture medium, fetal bovine serum and diabody.

6. the method for constructing the in-vitro GBM invasion model based on biological three-dimensional printing according to claim 1, wherein the GBM cell mass is obtained by the following steps:

Culturing U87 cells by adherence, centrifuging and blowing the cells uniformly by using a culture medium when the U87 cells adherent to the bottom of a culture bottle grow and proliferate to 90% of the volume, calculating the cell concentration, diluting to 100-200 ten thousand/ml, sucking 10 microliter of culture liquid drops containing U87 cells, dripping the culture liquid drops on the reverse side of a culture dish cover, adding 5 milliliters of PBS (phosphate buffer solution) into the culture dish to form a hydration environment cavity, finally covering the culture dish cover, wrapping the culture liquid drops by using a sealing film, and placing the culture liquid drops into a cell culture box at 37 ℃; the cells dispersed in the hanging drop are gradually aggregated under the action of gravity to form a plurality of flaky aggregates, the flaky aggregates gradually approach each other and are aggregated, and after 6-8 days of culture, spheres with the diameter of 200-300 mu m are finally formed, namely GBM cell masses.

Technical Field

The invention relates to a method for constructing an in-vitro GBM invasion model based on biological three-dimensional printing, which is used for constructing the in-vitro GBM invasion model with high cell survival rate and belongs to the fields of tissue engineering technology and biological three-dimensional (3D) printing.

Background

cancer seriously jeopardizes the life health and safety of people. The global disease burden organization research shows that only 2016 years of research show that 893 million people die due to cancer, and 2017 cancer progress reports issued by the american cancer research association (AACR) show that 2400 million cancer cases are likely to be broken through in 2035 because the number of cancer cases and the number of cancer deaths diagnosed every year in the world are increasing, which causes a great rise in medical cost, and 429 million new cases and 281 million deaths in 2015 in China are new cases, and a new method for more effective research and treatment of cancer is urgently needed.

It is well known that the development of anticancer drugs requires a great investment of labor, money and time, often costing several decades and several billion dollars to develop a practical and effective anticancer drug. Although the traditional method of two-dimensional planar cell culture is usually adopted in the anti-cancer drug test, the method is simple and fast, but the biological information contained in the microenvironment on the two-dimensional surface is far away from the real in-vivo extracellular matrix three-dimensional network containing various specific biological signals (biomechanics and cell signal transduction), because the perception, recognition, stimulus-response behavior and self-function regulation of the cells to the microenvironment have significant differences and can not reflect the real tumor cell behavior in vivo, the drug development failure is caused. In recent years, three-dimensional porous materials, hydrogels, and the like with excellent biocompatibility have been used to construct in vitro tumor models, and it has been found that tumor cells can grow, proliferate, migrate, and differentiate in a three-dimensional porous scaffold.

Gliomas, also known as glioblastoma multiforme (GBM), are the most aggressive of the brain-like tumors. It accounts for 15% of brain tumors, the second most common tumor in brain tumors, second only to meningiomas. The current treatments for it are mainly surgical resection, radiation therapy and chemotherapy, but even with the greatest degree of treatment, gliomas frequently recur, with patients usually having a life span of only 12 to 15 months after diagnosis, and only 3% to 5% of the patients can survive for more than five years. Survival time is usually only three months if not treated. Every one hundred thousand people per year are diagnosed with the disease. The biggest difference between GBM and other tumors is the existing in-situ invasiveness, and GBM does not carry out distant metastasis like other cancers but can be invaded in situ, so that GBM is easy to relapse after operation. Therefore, the invasion characteristics of the glioblastoma are researched, and the understanding of the molecular mechanism of occurrence and development of the glioblastoma is of great significance for treating the diseases. The traditional two-dimensional cell culture model lacks extracellular matrix and complex three-dimensional structure, and the animal model has high cost and ethical problems, so that a new research means for researching the invasive growth of GBM is urgently needed. The 3D biological printing technology can quickly and simply construct a three-dimensional hydrogel system containing cells, so that the direct construction of an in vitro 3D glioma model becomes possible. Most of the existing in vitro tumor models at home and abroad are latticed or solid models formed by hydrogel containing cells in the modes of extrusion, ink jet and the like, or a tumor microenvironment substrate is constructed by the hydrogel, and then the related cells such as tumors and the like are embedded or injected into the hydrogel environment.

Disclosure of Invention

Aiming at the defects of the prior art, the invention discloses a method for constructing an in-vitro GBM invasion model based on biological three-dimensional printing,

The technical scheme of the invention is as follows:

The method for constructing the in-vitro GBM invasion model based on biological three-dimensional printing is characterized by comprising the following steps of:

1) preparing biological ink containing human brain glial cells; the solvent of the biological ink is purified water, and the components of the biological ink comprise hyaluronic acid, alginate and gelatin; the content of the hyaluronic acid in the bio-ink is 0.01-0.02g/ml, the content of the alginate in the bio-ink is 0.01-0.02g/ml, and the content of the gelatin in the bio-ink is 0.05-0.15 g/ml;

2) Loading the biological ink into a feeding system of a multi-nozzle micro-extrusion biological three-dimensional printer, and printing a support structure according to a specified path; impregnating the obtained support structure with a calcium chloride solution;

3) Uniformly inoculating GBM cell masses into the scaffold structure obtained in the step 2);

4) Placing the support structure inoculated with the GBM into a cell culture box for continuous culture, changing the liquid every 2-3 days to obtain an in-vitro GBM invasion model with high cell survival rate, and respectively extracting RNA of the culture structure for sequencing on 1 st, 7 th and 14 th days of culture.

preferably, in the bio-ink, the selected content of the hyaluronic acid in the bio-ink is 0.02g/ml, the selected content of the alginate in the bio-ink is 0.02g/ml, and the selected content of the gelatin in the bio-ink is 0.010 g/ml.

Preferably, the bio-ink further comprises glucose as an invasion inducing factor, and the addition amount of the glucose is 0.005-0.02 g/ml.

Preferably, the support structure is a single-layer or multi-layer grid structure or a three-dimensional frame structure.

Preferably, in the step 4), the culture medium used in the culture process is prepared from a common cell culture medium, fetal bovine serum and double antibody.

Preferably, the method for obtaining the GBM cell mass adopts a hanging drop method, and comprises the following specific processes: culturing U87 cells by adherence, centrifuging and blowing the cells uniformly by using a culture medium when the U87 cells adherent to the bottom of a culture bottle grow and proliferate to 90% of the volume, calculating the cell concentration, diluting to 100-200 ten thousand/ml, sucking 10 microliter of culture liquid drops containing U87 cells, dripping the culture liquid drops on the reverse side of a culture dish cover, adding 5 milliliters of PBS (phosphate buffer solution) into the culture dish to form a hydration environment cavity, finally covering the culture dish cover, wrapping the culture liquid drops by using a sealing film, and placing the culture liquid drops into a cell culture box at 37 ℃; the cells dispersed in the hanging drop are gradually aggregated under the action of gravity to form a plurality of flaky aggregates, the flaky aggregates gradually approach each other and are aggregated, and after 6-8 days of culture, spheres with the diameter of 200-300 mu m are finally formed, namely GBM cell masses.

The invention can ensure that the survival rate of cells in the model reaches more than 90 percent and the mechanical property of the structure is kept stable through a composite regulation and control method.

Generally, in order to make the microenvironment constructed by the in vitro model as close to the real environment in vivo as possible so as to simulate various biological physiological processes such as in vivo drug reaction as truly as possible, the components of the biological ink are different according to different cells and tissues.

Here, since about 20% of the volume of the human brain is extracellular matrix, most of which is composed of Hyaluronic Acid (HA), and 90% of the cell volume of the brain is glial cells, a mixed system of HA and HEB is selected to simulate the composition of the brain. While the direct printing performance of bioprinted tissue structures may depend on the physical properties (e.g., viscosity) of the bio-ink itself, the subsequent stability of the structure relies on an additional crosslinking step. The invention selects a physical crosslinking mode of sodium alginate and calcium chloride solution (40 wt.%) by calcium ion gelatinization, and finely adjusts the mechanical properties of the hydrogel matrix obtained after bioprinting in a wide range including tissue rigidity and elasticity by changing the concentration and crosslinking density of the polymer forming the biological ink; gelatin is one of very important natural biological high molecular materials, has the functions of absorbing water and supporting a framework, and after being dissolved in water, gelatin particles can be mutually attracted and interwoven to form a laminated net structure and can be condensed along with the temperature reduction, so that a printing structure can keep a stable form and cannot deform even if a large load is borne. The structure brittleness can be reduced in the printing process, and the forming is facilitated. And gelatin is a natural biomaterial with excellent biological activity, such as biocompatibility and intrinsic cell adhesion ligands (e.g., RGD), which is beneficial to the survival of cells in the structure.

therefore, the correct selection of the components of the biological ink and the reasonable individualized process regulation and control according to various properties of the biological ink, such as viscosity and the like, in the printing process enable the survival rate of cells in the finally obtained GBM invasion model to reach more than 90 percent and the mechanical properties of the structure to be still stable after the cells are continuously cultured in a cell culture box at the constant temperature of 37 ℃ for 14 days.

The in vitro GBM invasion model obtained by the invention can be used for researching biological problems such as gene expression difference before and after invasion, the invasion characteristics of GBM under the action of different induced invasion factors and the like by simulating the in vivo GBM invasion process, and lays theoretical and experimental foundation for researching the molecular mechanism of glioma invasive growth. Meanwhile, the construction method of the model can also be suitable for other biological systems, and provides a new method and means for simulating the interaction between cells and controlling the microenvironment.

Induction of tumor cell migration and invasion is one of the important functions of in vitro tumor models. In most examples of two-dimensional culture research and chip construction, domestic and foreign scholars widely adopt a method for manufacturing a hypoxia or anoxic environment to induce tumor invasion, while in model construction of an in-vitro three-dimensional complex tissue organ, the design of a local hypoxia environment is difficult, and other introduced materials and local special structure design can also cause negative influence to a certain degree on the accuracy of a simulated in-vivo tumor microenvironment, so that glucose is used as an invasion inducing factor. The bottom of the model consists of a solid quadrangular film-like structure printed with bio-ink to which 0.005-0.02g/ml (preferably-0.02 g/ml) of glucose is added, serving as an induction of the invasive process of the tumor cells. The design of the concentration gradient formed by glucose diffusion to the upper part of the model can effectively induce the tumor cells in the GBM spheroids inoculated on the surface of the model to invade downwards so as to simulate the invasion process of glioma cells in vivo.

Drawings

FIG. 1 is a schematic diagram of the scaffold structure of an in vitro GBM invasion model.

FIG. 2 is a schematic diagram of the construction method of the in vitro GBM invasion model.

Detailed Description

The invention is further described below with reference to the accompanying drawings and examples.

As shown in fig. 1-2, the method for constructing an in-vitro GBM invasion model based on biological three-dimensional printing in this embodiment includes the following steps:

1) Preparing biological ink containing human brain glial cells; the solvent of the biological ink is purified water, and the components of the biological ink comprise hyaluronic acid, alginate and gelatin; the content of the hyaluronic acid in the biological ink is 0.02g/ml, the content of the alginate in the biological ink is 0.02g/ml, and the content of the gelatin in the biological ink is 0.010 g/ml. The bio-ink also comprises glucose as an invasion inducing factor, and the addition amount of the glucose is 0.02 g/ml.

2) loading the biological ink into a feeding system of a multi-nozzle micro-extrusion biological three-dimensional printer, and printing a support structure according to a specified path; the resulting scaffold structure was impregnated with calcium chloride solution as shown in figure 1;

3) Uniformly inoculating GBM cell masses into the scaffold structure obtained in the step 2);

4) placing the support structure inoculated with the GBM in a cell culture box for continuous culture, and changing the liquid every 2 days to obtain an in-vitro GBM invasion model with high cell survival rate.

To obtain GBM micro-tumor spheroids, the present invention uses the pendant drop method. When U87 cells attached to the wall at the bottom of a culture bottle grow and proliferate to about 90% of the volume, centrifuging, blowing and mixing uniformly by using a culture medium, calculating the cell concentration, diluting to 100-200 ten thousand/ml, sucking a plurality of culture liquid drops containing U87 cells of about 10 mu l, dripping the culture liquid drops on the reverse side of a culture dish cover with the diameter of 60mm, paying attention to a certain interval between every two drops to prevent the drops from touching each other, adding 5 ml of PBS (phosphate buffered saline) into the culture dish to form a hydration environment cavity, finally covering the culture dish cover, wrapping the culture dish cover by using a sealing film, and putting the culture dish into a cell culture box with the temperature of 37 ℃. The cells dispersed in the hanging drop are gradually gathered to form a plurality of flaky aggregates under the action of gravity, the flaky bodies gradually approach to each other and are polymerized, and after 6-8 days of culture, spheres with the diameter of about 200-300 mu m are finally formed, namely GBM micro-tumor spheroids.

The invention can be applied to the fields of drug screening and the like, and can research the molecular mechanism of the invasive growth of GBM and the molecular mechanism of the invasive growth of BM under the action of drugs by setting a plurality of groups of control experiments. The three-dimensional control group may include GBM spheroids cultured alone, GBM invasion models without human glial cells in scaffolds, and model scaffolds with human glial cells only without seeded GBM spheroids; the two-dimensional control group includes a control group which contains only one of glioma cell U87 and human brain glial cell HEB and is subjected to single two-dimensional culture, and a control group which is subjected to mixed two-dimensional culture of the two cells according to the same proportion in the experimental group. The models of the experimental group and the control group are cut into pieces on the 1 st, 7 th and 14 th days of culture, RNA of all cells in the models is extracted by adopting a standard RNA extraction step, RNA sequencing is carried out, and the sequencing result is analyzed. By comparing the results of gene sequencing, the difference of gene expression between the three-dimensional co-culture of various cells and the three-dimensional or two-dimensional culture of single cells can be obtained, and more effective experimental data are accumulated for researching the molecular mechanism of GBM invasive growth.

the planar tumor model has obvious difference with the in vivo environment, especially on drug response. This difference is due to the lack of a functional 3D tumor microenvironment by these two-dimensional models, resulting in insufficient interaction between cells and the extracellular matrix. Furthermore, during plastic surface culture (control group in two-dimensional culture alone), cells tend to show more phenotype that enables them to adhere to a rigid substrate. Thus, the availability and effectiveness of planar tumor models is limited. The in vitro GBM invasion model obtained by the invention can be used for researching biological problems such as gene expression difference before and after invasion, the invasion characteristics of GBM under the action of different induced invasion factors and the like by simulating the in vivo GBM invasion process, and lays theoretical and experimental foundation for researching the molecular mechanism of glioma invasive growth. Meanwhile, the construction method of the model can also be suitable for other biological systems, and provides a new method and means for simulating the interaction between cells and controlling the microenvironment.