CN110904044A

CN110904044A - Three-dimensional culture method of tumor stem cells

Info

Publication number: CN110904044A
Application number: CN201911299236.9A
Authority: CN
Inventors: 段新瑞; 汤薇
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2019-12-17
Filing date: 2019-12-17
Publication date: 2020-03-24
Anticipated expiration: 2039-12-17
Also published as: CN110904044B

Abstract

The invention discloses a three-dimensional culture method of tumor stem cells, which takes photosensitive resin as a raw material to print a honeycomb-shaped three-dimensional culture bracket in a 3D way, and H is contained₂S slow-release donor JK1 is electrospun at the bottom of the culture scaffold, growth factors are used for modifying the surface of the electrospun membrane to prepare a three-dimensional culture scaffold, and then the scaffold is used for culturing tumor stem cells. The photosensitive resin is selected as the material of the 3D printing support, and the material has good biocompatibility and high mechanical strength; regulating pH to H₂Incorporation of S donor JK1 into electrospinning and modification of broth pH for H₂The release of S is regulated and controlled so as to regulate the growth speed of cells; the growth factor is modified on the surface of the electrospinning fiber to promote the proliferation of the tumor stem cells. The invention utilizes the film-covered honeycomb-shaped culture bracket to culture the tumor stem cells with high quality and high efficiency, effectively promotes the proliferation of the tumor stem cells, simultaneously keeps the dryness of the tumor stem cells and avoids differentiation, and lays a foundation for the characteristic research of the tumor stem cells and the development of targeted drugs.

Description

Three-dimensional culture method of tumor stem cells

Technical Field

The invention belongs to the technical field of stem cell culture, and particularly relates to a three-dimensional culture method of novel tumor stem cells.

Background

Tumor stem cells (CSCs) are a small fraction of the tumor cell population, have the ability to promote tumorigenesis, maintain tumor growth, and maintain tumor heterogeneity, are capable of self-renewal, multi-differentiation, and immortalization, and express stem cell surface markers. Research has shown that CSCs have tumor cell regenerative capacity and contribute to tumor heterogeneity, multidrug resistance and radioresistance, as well as early micrometastases. Therefore, CSCs are closely associated with metastasis, recurrence and drug resistance of tumors, and research and development of drugs and therapies against CSCs are crucial to improve the cure rate of cancer and reduce the tumor recurrence rate. However, because the number of CSCs in the tumor cell population is very small and most CSCs are in the stationary phase, it is difficult to achieve a large amount of amplification of CSCs in vitro using the conventional two-dimensional culture method, and thus the requirement for CSCs research at present cannot be met, and it is difficult to establish an in vitro drug model for CSCs. The most common method for culturing CSCs in vitro is serum-free suspension culture. The culture method enables the CSCs to grow in suspension in a culture solution without serum and added with high-concentration growth factors to grow in a spherical shape. Although a certain amount of CSCs can be obtained by the method, the culture time is long, the cell proliferation efficiency is low, and the cell balls are easy to contact with each other to form large cell masses to induce cell differentiation, so that the stem cell proportion is low.

The three-dimensional cell culture technology is a simple and effective cell culture mode developed on the basis of two-dimensional cell culture and animal experiment models. Compared with two-dimensional culture, the three-dimensional culture can accommodate high-density cell adhesion and proliferation, is favorable for intercellular signal transmission, and provides a proper microenvironment for the metabolic activity of the maintenance cells. In addition, the three-dimensional culture can increase the content of extracellular matrix, and the activity and the function of the cultured cells are enhanced. The in vivo microenvironment can be better simulated by utilizing the three-dimensional cell culture technology, so that the inhibition effect of the drug on the tumor can be better researched.

Disclosure of Invention

The invention aims to solve the problems of slow proliferation speed and easy clustering and differentiation of tumor stem cells in-vitro culture, and provides a novel three-dimensional culture method of the tumor stem cells.

Aiming at the purposes, the technical scheme adopted by the invention comprises the following steps:

1. the method comprises the following steps of printing a honeycomb culture support by using an LCD (liquid crystal display) photocuring 3D printer by using acrylic resin as a raw material, wherein the honeycomb culture support consists of a honeycomb cell culture support and a fixed support.

2. Adding Polycaprolactone (PCL) into a mixed solution of chloroform and methanol in a volume ratio of 6-8: 1, and stirring at normal temperature for 10-12H, then adding H₂S, releasing a donor JK1, continuously stirring for 10-12 h, adding fibroin, and uniformly stirring to obtain a spinning solution; the concentration of polycaprolactone in the spinning solution is 0.1-0.3 g/mL, and the mass ratio of polycaprolactone to JK1 to fibroin is 1: 0.01-0.2: 0.1-0.3.

The structural formula of the above JK1 is shown below:

it is prepared according to the method in the literature "pH-controlledhydrogensulfate release for myocarpidischemia-reperfusion in jury, J.Am.chem.Soc., 2016".

3. And (3) spinning at the bottom of the honeycomb cell culture rack in the step 1 by using the spinning solution in the step 2 to form a polycaprolactone/JK 1 composite fiber membrane.

4. And (3) modifying the polycaprolactone/JK 1 composite fiber membrane in the step (3) by using Growth Factors (GF) by adopting a layer-by-layer self-assembly technology.

5. Fixing the honeycomb cell culture frame modified in the step 4 by using a fixing support, placing the fixed honeycomb cell culture frame into a culture dish, resuspending the collected tumor stem cells by using a serum-free culture medium, adding the suspended tumor stem cells into the culture dish, slightly shaking the culture dish to enable the cells to sink into the honeycomb cell, and performing conventional culture.

In the step 1, the honeycomb cell culture rack is preferably circular, the height of the honeycomb cells is 0.2-1 mm, and the side length of the honeycomb cells is 0.1-0.5 mm.

In the step 2, the concentration of polycaprolactone in the spinning solution is preferably 0.15-0.2 g/mL, and the mass ratio of polycaprolactone to JK-1 to fibroin is 1: 0.05-0.1: 0.1-0.3.

In the step 3, the diameter of the fiber of the polycaprolactone/JK-1 composite fiber membrane is preferably 0.5-1.5 μm.

In the step 4, the growth factors are any two of leukemia inhibitory factor, platelet-derived growth factor, insulin-like growth factor, recombinant human epidermal growth factor and recombinant human basic fibroblast, and preferably recombinant human epidermal growth factor and recombinant human basic fibroblast.

The invention has the following beneficial effects:

1. the stem cell culture method provided by the invention can obviously improve the balling rate of the tumor stem cells, promote cell proliferation, improve the proportion of the stem cells and keep the dryness of the cells, thereby providing a foundation for the research of medicaments and cytology related to the tumor stem cells.

The 2.3D printed honeycomb-shaped small chamber has fine structure, is beneficial to the growth of the tumor stem cells in a sphere shape, and avoids the differentiation induced by the contact fusion between the cell spheres.

3. The polycaprolactone/JK 1 composite fiber membrane modified with growth factors provides a three-dimensional environment for cell growth, wherein H released by JK1₂S and growth factors promote the proliferation of tumor stem cells. In addition, since JK1 is sensitive to pH conditions, H can be controlled by changing pH conditions₂The release speed of S, and further the proliferation speed of the tumor stem cells is regulated and controlled.

4. Can change honeycomb culture support size according to the culture dish size, support part repeatedly usable, convenient to use, the practicality is strong.

Drawings

FIG. 1 is a photograph of a honeycomb culture support in example 1.

Fig. 2 is a FTIR spectrum of polycaprolactone/JK 1 composite fiber film 1 formed by JK1, PCL and different incorporation amounts of JK 1.

FIG. 3 is a scanning electron microscope image of polycaprolactone/JK 1 composite fiber films formed by PCL and JK1 with different doping amounts.

FIG. 4 is H of JK1₂S release profile.

FIG. 5 is H of polycaprolactone/JK 1 composite fiber film₂S release curve (right).

FIG. 6 is a comparison of the numbers of tumor cells cultured in the culture method of example 1 (experimental group) and in the conventional culture method (control group).

FIG. 7 is a comparison of the number of cells cultured in the culture method of example 1 (experimental group) and the conventional culture method (control group).

Detailed Description

The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.

Example 1

1. A honeycomb support model is manufactured by applying FreeCAD design software, acrylic resin is injected into an LCD photocuring 3D printer and is printed with a honeycomb culture support, the curing wavelength is 405nm, the structure of the honeycomb culture support is shown in figure 1a and consists of a honeycomb cell culture frame (left) and a fixed support (right), the honeycomb cell culture frame is circular, the diameter of the honeycomb cell culture frame is 18mm, and the height of the honeycomb cell culture frame is 1mm and the side length of the honeycomb cell culture frame is 0.5 mm.

2. 0.4800g of polycaprolactone is added into 3.2mL of mixed solution of chloroform and methanol with the volume ratio of 7:1, the mixed solution is stirred for 12 hours at normal temperature, then 0.0480g of JK1 is added, the stirring is continued for 12 hours, then 0.096g of fibroin is added, and the stirring is uniform, so that the spinning solution is obtained. The concentration of polycaprolactone in the spinning solution is 0.15g/mL, and the mass ratio of polycaprolactone to JK1 to fibroin is 1:0.1: 0.2.

3. Sucking 1mL of the spinning solution obtained in the step 2 by using a glass syringe, connecting the glass syringe filled with the spinning solution with a stainless steel needle, then placing the syringe on an injection pump, wherein a receiving device is a metal copper plate with aluminum foil, and placing the bottom of the honeycomb cell culture rack obtained in the step 1 upwards in the center of the metal copper plate. Adjusting the speed of an injection pump to be 2mL/h, controlling a high-voltage power supply to be 6-7 KV, controlling the distance between a needle of an injector and a metal copper plate to be about 10.0cm, and carrying out electrostatic spinning at room temperature. After spinning, the injection pump and the high-voltage power supply are disconnected, the honeycomb cell culture rack is taken out, and at the moment, the polycaprolactone/JK 1 composite fiber membrane (marked as PCL-JK 1-10%) is attached to the bottom of the honeycomb cell culture rack.

4. Dissolving chitosan in 2% acetic acid water solution (pH 4.5) to prepare chitosan solution with concentration of 1 mg/mL; recombinant human basic fibroblast growth factor (bFGF) is added into the chitosan solution to ensure that the concentration of the bFGF is 10 mu g/mL, and then the chitosan/bFGF solution is prepared. Recombinant human Epidermal Growth Factor (EGF) was dissolved in PBS buffer (0.1% BSA, pH 7.2) to prepare an EGF solution having a concentration of 10 μ g/mL. Immersing the film-coated honeycomb cell culture rack spun in the step 3 in a chitosan/bFGF solution for 15min, taking out, washing 3 times for 3min each with a 1mg/mL NaCl aqueous solution to remove the redundant solution, then immersing again in an EGF solution for 15min, taking out, washing 3 times for 3min each with a NaCl solution (1mg/mL), and circulating in this way. And (3) one layer is modified by immersing the substrate in a chitosan/bFGF solution and an EGF solution at one time, 20 layers are modified totally, and the outermost layer is modified into EGF.

5. Drying the modified honeycomb cell culture frame in the step 4 at 25 ℃ for 24h, irradiating and sterilizing the modified honeycomb cell culture frame by using Co60 overnight, fixing the modified honeycomb cell culture frame by using a fixing frame (see figure 1B), placing the modified honeycomb cell culture frame into a culture dish, digesting and centrifuging the HCT116 cells which are cultured conventionally, then re-suspending the cells by using a serum-free culture medium (consisting of a DMEM/F12 culture medium and a 2% B27 additive) to prepare a single cell suspension, adding the single cell suspension into the culture dish, slightly shaking the culture dish to enable the cells to sink into the honeycomb cell, and culturing the cells in a cell culture box.

Example 2

In step 2 of this example, 0.4800g of polycaprolactone is added to 3.2mL of a mixed solution of chloroform and methanol at a volume ratio of 7:1, stirred at room temperature for 12 hours, then 0.00480g of JK1 is added, stirring is continued for 12 hours, then 0.096g of fibroin is added, and stirring is performed uniformly, so as to obtain a spinning solution. The concentration of polycaprolactone in the spinning solution is 0.15g/mL, and the mass ratio of polycaprolactone to JK1 to fibroin is 1:0.01: 0.2. After the spinning solution is used for electrostatic spinning according to the method of the step 3 in the embodiment 1, a polycaprolactone/JK 1 composite fibrous membrane is attached to the bottom of the honeycomb cell culture rack and is marked as PCL-JK 1-1%. The other steps are the same as in example 1.

Example 3

In step 2 of this example, 0.4800g of polycaprolactone was added to 3.2mL of a mixed solution of chloroform and methanol at a volume ratio of 7:1, stirred at room temperature for 12 hours, then 0.0240g of JK1 was added, stirring was continued for 12 hours, then 0.096g of fibroin was added, and stirring was performed uniformly to obtain a spinning solution. The concentration of polycaprolactone in the spinning solution is 0.15g/mL, and the mass ratio of polycaprolactone to JK1 to fibroin is 1:0.05: 0.2. After the spinning solution is used for electrostatic spinning according to the method of the step 3 in the embodiment 1, a polycaprolactone/JK 1 composite fibrous membrane is attached to the bottom of the honeycomb cell culture rack and is marked as PCL-JK 1-5%. The other steps are the same as in example 1.

Example 4

In step 2 of this example, 0.4800g of polycaprolactone was added to 4.0mL of a mixed solution of chloroform and methanol at a volume ratio of 7:1, stirred at room temperature for 12 hours, then 0.0240g of JK1 was added, stirring was continued for 12 hours, then 0.096g of fibroin was added, and stirring was performed uniformly to obtain a spinning solution. The concentration of polycaprolactone in the spinning solution is 0.12g/mL, and the mass ratio of polycaprolactone to JK1 to fibroin is 1:0.01: 0.2. The other steps are the same as in example 1.

Example 5

In step 2 of this example, 0.4800g of polycaprolactone was added to 2.7mL of a mixed solution of chloroform and methanol at a volume ratio of 7:1, stirred at room temperature for 12 hours, then 0.0240g of JK1 was added, stirring was continued for 12 hours, then 0.096g of fibroin was added, and stirring was performed uniformly to obtain a spinning solution. The concentration of polycaprolactone in the spinning solution is 0.18g/mL, and the mass ratio of polycaprolactone to JK1 to fibroin is 1:0.01: 0.2. The other steps are the same as in example 1.

The polycaprolactone/JK 1 composite fiber membranes obtained in the above examples were characterized, and the tumor cells cultured in the manner of example 1 were subjected to cell proliferation and desiccation experiments, the experimental methods and results were as follows:

1. characterization of polycaprolactone/JK 1 composite fiber film

The structure identification of the polycaprolactone/JK 1 composite fiber film formed by the JK1 and the PCL alone and the JK1 with different doping amounts in the examples 1-3 is carried out by a Fourier mid-infrared-far infrared spectrometer, and the result is shown in figure 2. As shown in the figure, the absorption frequency indicated by the dotted line is 3320cm^-1Is the N-H stretching vibration of JK 1; 1600cm^-1Is the N-H bending vibration of JK 1; 720cm^-1Is the C-H out-of-plane bending vibration on the aromatic ring and indicates that the aromatic ring is monosubstituted. As can be seen from the above infrared analysis, JK1 has been incorporated into PCL fiber films.

The morphology of the polycaprolactone/JK 1 composite fibrous membrane formed by PCL and JK1 with different doping amounts is characterized by a desk type scanning electron microscope. As shown in FIG. 3, the diameter of the fiber of the polycaprolactone/JK 1 composite fiber film is about 0.8-1.0 μm, the fiber film has a uniform morphology and a smooth surface, no crystal is observed, which indicates that the loaded H is₂JK1 of the S donor was evenly distributed on the fiber membrane. As can be seen by analysis, the diameter of the fiber film shows a gradually decreasing trend along with the increase of the concentration of the doped donor JK1, namely PCL (a)>PCL-JK1-1％(b)>PCL-JK1-5％(c)>PCL-JK1-10％(d)。

For H under different pH conditions₂The release profile of S was recorded. JK1 and polycaprolactone/JK 1 composite fiber membranes are respectively placed in 50mL of 50nmol/L PBS buffer solution under different pH conditions (pH 7.4, pH 6.8 and pH 6.0), 2mL of solutions are respectively taken out at set time points, and 200 mu L of 0.01mg/mL zinc acetate aqueous solution and 400 mu L of 30mmol/L FeCl are sequentially added₃The solution (prepared from 1.2mol/L hydrochloric acid solution) and 400. mu.L of 20mmol/L N, N-dimethyl-1, 4-phenylenediamine hydrochloride solution (prepared from 7.2mol/L hydrochloric acid solution) were reacted for 20 minutes, and then the ultraviolet absorption at 670nm was measured. The results are shown in FIGS. 4 and 5, H released by JK1₂The S concentration reached a maximum at 40 minutes and then began to slowly decrease. H is generated in a polycaprolactone/JK 1 composite fiber membrane formed by electrostatic spinning of spinning solutions with polycaprolactone content of 0.12g/mL, 0.15g/mL and 0.18g/mL₂The peak time of S is 3.5 hours, 3.5 hours and 4.5 hours respectively, and is 5-6 times longer than the release time of JK1 alone. These results indicate that the polycaprolactone/JK 1 composite fiber membrane significantly prolongs H₂The release time of S plays a role in slow release.

2. Experiment on proliferation ability and cell dryness of tumor stem cells

The normally cultured tumor cells were collected by centrifugation, resuspended in serum-free medium, and adjusted to a cell density of 5000 cells/mL. The culture method of example 1 was used as an experimental group, and a coated honeycomb-shaped culture scaffold was placed in each well of a six-well plate, and then a cell suspension was added to the well plate at 2mL per well and cultured at 37 ℃. As a control group, a six-well plate was plated with 1% agarose gel, and the cell suspension was added to the six-well plate at 37 ℃ in 2mL per well. After 9 days of culture, the tumor stem cells obtained in example 1 and the conventional culture method were examined, respectively. The results are shown in fig. 6, where the number of stem cell-like tumor microspheres in the experimental group is significantly greater than that in the control group. The growth of tumor stem cells in the form of spheres is one of the characteristics of maintaining the cell dryness, and the results show that the culture method of example 1 can maintain the dryness of tumor stem cells well. Next, as a result of counting cells after digesting the cells as shown in fig. 7, the number of cells in the experimental group was significantly greater than that in the control group (p < 0.01). This result demonstrates that the culture method of example 1 can significantly promote the proliferation of tumor stem cells, compared to the conventional culture method.

Claims

1. A three-dimensional culture method of tumor stem cells is characterized in that:

(1) printing a honeycomb culture support by using an LCD (liquid crystal display) photocuring 3D printer, wherein the honeycomb culture support consists of a honeycomb cell culture support and a fixed support;

(2) adding polycaprolactone into a mixed solution of chloroform and methanol in a volume ratio of 6-8: 1, stirring at normal temperature for 10-12 hours, adding JK1, continuing stirring for 10-12 hours, adding fibroin, and uniformly stirring to obtain a spinning solution; the concentration of polycaprolactone in the spinning solution is 0.1-0.3 g/mL, the mass ratio of the polycaprolactone to JK1 to fibroin is 1: 0.01-0.2: 0.1-0.3, and the structural formula of JK1 is as follows:

(3) spinning at the bottom of the honeycomb cell culture rack in the step (1) by adopting the spinning solution in the step (2) to form a polycaprolactone/JK 1 composite fiber membrane;

(4) modifying the polycaprolactone/JK 1 composite fiber membrane in the step (3) by using growth factors by adopting a layer-by-layer self-assembly technology;

(5) fixing the honeycomb cell culture frame modified in the step (4) by using a fixing support, placing the fixed honeycomb cell culture frame into a culture dish, resuspending the collected tumor cells by using a serum-free culture medium, adding the suspended tumor cells into the culture dish, slightly shaking the culture dish to enable the cells to sink into the honeycomb cell, and performing conventional culture.

2. The method for three-dimensional culture of tumor stem cells according to claim 1, wherein: in the step (1), the honeycomb cell culture rack is circular, the height of the honeycomb cell is 0.2-1 mm, and the side length of the honeycomb cell is 0.1-0.5 mm.

3. The method for three-dimensional culture of tumor stem cells according to claim 1, wherein: in the step (2), the concentration of polycaprolactone in the spinning solution is 0.15-0.2 g/mL, and the mass ratio of polycaprolactone to JK1 to fibroin is 1: 0.05-0.1: 0.1-0.3.

4. The method for three-dimensional culture of tumor stem cells according to claim 1, wherein: in the step (3), the diameter of the fiber of the polycaprolactone/JK 1 composite fiber membrane is 0.5-1.5 μm.

5. The method for three-dimensional culture of tumor stem cells according to claim 1, wherein: in the step (4), the growth factors are any two of leukemia inhibitory factor, platelet-derived growth factor, insulin-like growth factor, recombinant human epidermal growth factor and recombinant human basic fibroblast.

6. The method for three-dimensional culture of tumor stem cells according to claim 5, wherein: the growth factor is recombinant human epidermal growth factor and recombinant human basic fiber.