KR101449906B1 - Biomimetic three dimensional cell culture method using agarose, collagen and alginate composite hydrogel scaffold - Google Patents

Abstract

The present invention relates to a method for culturing a three-dimensional cell using an agarose-collagen-alginate composite hydrogel scaffold. More specifically, the method comprises the steps of: mixing and culturing a cell in an alginate solution; preventing the separation of the cell by adding a collagen solution in the cultured mixture; and adding and stationing an agarose solution in the mixture including the collagen in order to be gelated. In the method of the present invention, a cell culturing environment which is very similar to the human body is composed, and spheroid and spheroid budding forming functions, cell survival function, growing functions, proliferating functions, differentiating functions, and drug diffusing functions are excellent. Accordingly, the method of the present invention can be effectively used in studying the formation, growth, transition, and death of cancer cells, testing drug sensitivity, diagnosing cancer, discerning treatment effects, developing a technique for predicting prognoses, and developing a treating agent having excellent biocompatibility.

Description

[0002] Biomimetic three-dimensional cell culture method using agarose, collagen and alginate composite hydrogel scaffold [

The present invention relates to a three-dimensional cell culture method for biomimetry using a complex hydrogel support containing agarose, collagen and alginate as an active ingredient, and a use thereof.

Cell culture is the most fundamental research method in the field of biotechnology, and it is used extensively in researching not only biological functions but also human diseases. Although 40 years have passed since the development of cell culture methods for eukaryotic cells, the most commonly used method to support adherent cell growth is polystyrene or glass Lt; RTI ID = 0.0 > 2-dimensional < / RTI >

However, the cells grown by the two-dimensional cell culture method, which is a single cell culture method, are attached to the extracellular matrix and show many differences from the cells growing in the three-dimensional biotissue environment. Therefore, the two-dimensional and three-dimensional cell cultures show general morphological differences, and the expression of the receptor, gene transcription, cell migration and apoptosis, which occur through conventional two-dimensional cell culture, Since the complex life phenomenon differs greatly from the phenomenon actually occurring in the living tissue environment, the two-dimensional cell culture method has a problem that it can not accurately reflect the physiological environment of the living body in which the cells grow on the three-dimensional plane.

In addition, existing two-dimensional cell cultures mainly cultivate one kind of the same cells, so that different types of intercellular interactions can not be considered. Although some attempts have been made to solve this problem partially through 2D co-culture, there have been many differences from the cellular environment in living tissues.

Therefore, in order to solve the problem of two-dimensional cell culture, research and development of a three-dimensional cell culture method considering the spatial organization of cells have a very important significance.

Three-Dimensional Biological Tissue Simulation The main method used to construct the cell culture environment is to cultivate cells using a porous biocompatible scaffold. Natural polymers that are widely used as materials for producing hydrogels, which are the main forms of biocompatible scaffolds, include proteins such as collagen, gelatin, silk and fibrin, and polysaccharides such as alginate, agarose and chitosan. These natural polymers are separated from natural materials and widely used as clinical materials.

Many efforts have been made to develop various hydrogel supports for three-dimensional cell cultures using single or composite materials of natural polymers. However, there is a problem in that the efficiency of three-dimensional cell culture is not high due to the properties of each substance . For dihydro alginate most widely used as a material of the gel support, mainly by being cross-linked by other rare or non-2 Ca ^{2 +} cations by the Ca ^{+ 2} form a hydrogel. However, such ions have a problem that they show toxicity to cells, and since collagen has a low level of mechanical properties, there is a problem that gelation is difficult with collagen alone. In addition, agarose can be gelated alone and is used as a solid cell culture medium, but shows a problem of low cell culture efficiency.

Therefore, it is necessary to study and develop an effective three-dimensional cell culture method considering the spatial organization of cells by solving the problem of the hydrogel support of such a natural polymer material.

Korean Patent No. 10-0761922

In order to solve the above-mentioned problems, the present invention relates to a three-dimensional cell culture technique using a hydrogel support specially manufactured as a composite material of agarose, collagen and alginate, which is widely used as a natural polymer material, Collagen-alginate complex hydrogel support capable of solving the above problems, and to provide a three-dimensional cell culture method having excellent cell culture efficiency and its use.

The present invention relates to a method for producing an alginate solution, Adding a collagen solution to the cultured mixture to prevent cell detachment; And adding an agarose solution to the collagen-added mixture, and allowing the agarose solution to stand to gel.

The present invention relates to a method for producing an alginate solution, Adding a collagen solution to the cultured mixture to prevent cell detachment; Adding an agarose solution to the collagen-containing mixture, allowing the mixture to stand and gel, and culturing the cells three-dimensionally; And analyzing the growth, proliferation, death, differentiation and migration of the three-dimensional cultured cells.

The present invention also relates to a method for producing an alginate solution, Adding a collagen solution to the cultured mixture to prevent cell detachment; Adding an agarose solution to the collagen-containing mixture, allowing the mixture to stand and gel, and culturing the cells three-dimensionally; And analyzing the cell survival rate by adding an anticancer agent to the three-dimensional cultured cells.

The agarose-collagen-alginate complex hydrogel support according to the present invention solves the problem of cytotoxicity due to chemical crosslinking by inducing gelation by a physical method without chemical cross-linking using existing ions and provides high transparency, Cell observation is easy. In addition, the agarose-collagen-alginate complex hydrogel of the present invention has the same cell growth effect as the complex hydrogel support using the existing animal collagen using marine-derived collagen (hereinafter referred to as "marine collagen") and is more economical than animal collagen The excellent, high cell affinity, biocompatibility and low cytotoxicity can provide a safe and efficient cell culture method for cells during cell culture.

In addition, the three-dimensional cell culture method using the agarose-collagen-alginate complex hydrogel support of the present invention provides a cell culture environment very similar to that of a living body and has excellent spore forming ability, drug diffusing ability and cell growth ability. Therefore, the biomimetic three-dimensional cell culture method using the complex hydrogel support according to the present invention can be used for biological activity analysis through observation of cell growth, proliferation, death, differentiation and migration. In addition, it can be used for cancer development, malignant transformation, invasion and metastatic mechanism research, susceptibility to anticancer drugs and cancer prognosis analysis or evaluation, and can also be effectively used for the development of therapeutic agents having excellent bioavailability.

1 is a schematic view showing the composition of an agarose-collagen-alginate complex hydrogel support according to the present invention.
FIG. 2 is a schematic view showing a process for producing an agarose-collagen-alginate complex hydrogel support according to the present invention.
FIG. 3 shows the results of treatment of various concentrations of CaCl ₂ (10, 50, 100, 200 and 300 mM) for 18-24 hours and the cytotoxic effect of CaCl ₂ on CREC and murine cancer cells EL4, R182 and A2780 This is the result of the confirmed WST-1 analysis.
FIG. 4 is a graph showing the results of measurement of agarose-alginate complex hydrogel (hereinafter referred to as " 0.125% ") having a final concentration of agarose of 0.125% by weight and a final concentration of alginate of 1% by weight in a mixed solution of hydrogel and agarose- Weight% agarose-1 wt% alginate complex hydrogel ") suppository, respectively, and comparing the three-dimensional cell growth environment.
FIG. 5 is a phase contrast microscope photograph showing the growth of EL4 cells after three-dimensional culture of EL4 cells for 2, 5, and 10 days in agarose alone hydrogel at 0.5, 1, 1.5, and 2 wt% concentrations.
FIGS. 6 and 7 are graphs showing the results of a composite hydrogel support prepared by adding rat collagen (RC), an animal collagen, to a 0.125% agarose-1% by weight alginate mixture to a final concentration of 0.05% EL4 cells were three-dimensionally cultured on a complex hydrogel support in which marine collagen was added to agarose-1% by weight alginate solution so as to have final concentrations of 0.05, 0.1, 0.2, 0.5, 1, FIG. 6 is a photograph of a phase contrast microscope confirming the desorbed cells, and FIG. 7 is a graph showing the number of cells desorbed outside the respective hydrogel supports.
FIG. 8 is a graph showing the results of comparing the effects of marine collagen firstly tried in the present invention and animal collagen widely used as collagen contained in 0.125 wt% agarose-collagen-1 wt% alginate complex hydrogel support. EL4 cells were cultured in a three-dimensional environment on a collagen-containing supporter, and then cell growth was confirmed using a phase contrast microscope.
9 is a graph showing the results of comparison of the effect of marine collagen in the present invention with the animal collagen generally used as collagen contained in 0.125 wt% agarose-collagen-1 wt% alginate complex hydrogel support. A2780 cells were cultured in a three-dimensional environment in a supernatant containing a polyclonal antibody, and cell growth was confirmed by a phase contrast microscope.
10 is a phase contrast microscope photograph showing transparency of the agarose-collagen-alginate complex hydrogel support according to the present invention.
Figure 11 is a graph showing the results of a method of preparing an agarose gel composition comprising 1 wt% alginate alone, 0.125 wt% agarose alone, 7.5 or 10 wt% marine collagen alone hydrogel support, 0.125 wt% agarose-1 wt% alginate complex hydrogel support and 0.125 wt% agarose- 1 is a photograph showing a cross section of a marine collagen-1 wt% alginate and 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate composite hydrogel support observed with a scanning electron microscope (SEM).
FIG. 12 is a graph showing the results of an experiment in which 1 wt% alginate alone, 0.125 wt% agarose alone, 7.5 or 10 wt% marine collagen alone hydrogel support, 0.125 wt% agarose-1 wt% alginate complex hydrogel support, 0.125 wt% agarose- IR analysis on the weight% marine collagen-1 wt% alginate and 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel supports.
Fig. 13 is a schematic diagram of the molecular structure of each hydrogel material, Fig. 13A is a molecular structure diagram of agarose, Fig. 13B is a molecular structure diagram of marine collagen, and Fig. 13C is a molecular structure diagram of alginate.
14 is a graph showing the results of a new 0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate and 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel support Of the present invention.
FIG. 15 is a graph showing the results obtained by culturing human ovarian cancer cells in a 0.125% agarose-7.5% by weight marine collagen-1% by weight alginate complex hydrogel support according to the present invention for 7 days, (b), DAPI staining (c), and F-actin staining (d).
FIG. 16 is a graph showing the results of EL4 cells incubated for 1, 3, and 5 days in a 0.125% agarose-10% by weight marine collagen-1% by weight alginate complex hydrogel support according to the present invention and observing the formed cell spheroids with a phase contrast microscope Results.
FIG. 17 is a graph showing the results obtained by observing the spheroide burring phenomenon which is important for the formation of the cell sphere after culturing A2780 cells in 0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate complex hydrogel support according to the present invention As a result, FIG. 17A shows the results of the phase contrast microscope observation and FIG. 17B shows the H & E staining results.
18 shows the results of confirming the diffusion ability of a drug reaching cancer cells in a three-dimensional environment. As a result, it was confirmed that human ovarian cancer cell A2780 using 0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate complex hydrogel support The results are as follows.
19 is a graph showing the results of a three-dimensional culture using 0.127 wt% agarose-7.5 wt% marine collagen-1 wt% alginate complex hydrogel support according to the present invention and A2780, a human ovarian cancer cell, 5, and 7 days, respectively.
FIG. 20 is a CFSE staining result showing cell proliferation after three-dimensional culture of CREC and EL4 cells in a 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel support according to the present invention.
21 and 22 show that the agarose-marine collagen-alginate complex hydrogel support according to the present invention exhibited cytotoxicity upon cell culture. As a result, FIG. 21 shows that 0.125% agarose-7.5% by weight marine collagen- FIG. 22 shows the result of confirming Live / Dead cells by a confocal laser microscope after seeding A2780, a human ovarian cancer cell, on a weight% alginate complex hydrogel support and culturing for 7 and 10 days, The results of confirming whether Live / Dead cells were observed by a confocal laser microscope to determine whether cytotoxicity is shown by seeding CREC and EL4 cells on a 1% by weight marine collagen-1 wt% alginate complex hydrogel support and culturing for 14 days.
23 shows the results of three-dimensionally culturing EL4 cells in a two-dimensional culture and 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel support according to the present invention, The expression level of Notch was confirmed by RT-PCR analysis.
24 shows the results of three-dimensional culture of human ovarian cancer cell line A2780 cells in a two-dimensional culture and 0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate complex hydrogel support according to the present invention, CD44, an important marker of marker, snail and vimentin and cancer stem cell, the expression of endothelin-1, which plays a key role in tumor growth and metastasis .
25 shows the results of two-dimensional culture and culturing of lymphoma cells (EL4) and human ovarian cancer cells (A2780) in a three-dimensional environment in a 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel support according to the present invention Cell survival rate after chemotherapy.
26 is a graph showing the results of three-dimensional culture of human ovarian cancer cells (A2780) in a two-dimensional and 0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate complex hydrogel support according to the present invention, Paclitaxel, and curcumin, which have anti-cancer effects against various cancer cells.
FIG. 27 is a graph showing the results of cultivating human ovarian cancer cells (A2780) in a three-dimensional environment in a 0.125% by weight agarose-7.5% by weight marine collagen-1% by weight alginate complex hydrogel support according to the present invention Immunostaining with antibodies against cytokeratin, a marker, was followed by phase contrast microscopy and fluorescence microscopy.
28 is a flow cytometric analysis result of measurement of cancer stem cell-forming ability and cancer cell differentiation ability on EL4 cells cultured three-dimensionally in 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel support.

The present invention relates to a method for producing an alginate solution, Adding a collagen solution to the cultured mixture to prevent cell detachment; And adding an agarose solution to the collagen-added mixture, and allowing the agarose solution to stand to gel.

More particularly, the three-dimensional cell culture method comprises: mixing an alginate solution and cells and culturing at a temperature of 25 to 37 ° C for 5 to 30 minutes; Adding a collagen solution to the cultured mixture to prevent desorption of the cells at a temperature of 25 to 37 DEG C for 5 to 30 minutes; And adding the agarose solution to the collagen-added mixture and allowing the gelatin to stand for 5 to 30 minutes at a temperature of 4 to 25 ° C.

In the step of mixing and culturing the alginate solution and the cells, the incubation temperature and time range are the most preferable temperature and time range for minimizing the effect on the cells.

When the collagen solution is added to the cultured mixture to prevent cell detachment, the collagen is not homogeneously mixed or the cell culture efficiency is lowered if the temperature range or the time range when the collagen solution is added is out of the range Can be caused.

In addition, when the agarose solution is added and allowed to stand and gelated, if the temperature is out of the temperature range or time range, gelling may not be performed or cells may be damaged or killed.

The collagen may be marine-derived collagen, but is not limited thereto.

The alginate may be added in an amount of 0.5 to 2 parts by weight, more preferably 1 part by weight, based on 100 parts by weight of the mixture, but is not limited thereto.

The collagen may be added in an amount of 1 to 30 parts by weight, more preferably 7.5 to 10 parts by weight, based on 100 parts by weight of the mixture, but is not limited thereto.

The collagen is a fibrous protein, which is a natural polymer that constitutes the main component of the outer cell matrix in the connective tissue. Collagen acts as a regulator of cell recognition and cell attachment and proliferation. It supports the tissues and organs, maintains the body shape surrounding the body tissues, and plays a role in tissue regeneration. It is composed of sponge, fiber form, gel, solution, It is possible to process various shapes such as tubular material. However, it is difficult to control the mechanical strength and decomposition rate of collagen at a very low level, and it is difficult to gel by using alone collagen. Therefore, if the collagen solution is out of the above-mentioned content range, the cells may not be encapsulated so that the cells may escape from the gel or affect the growth ability of the cells.

Agarose may be added in an amount of 0.1 to 0.5 parts by weight, more preferably 0.125 parts by weight, based on 100 parts by weight of the mixture, but is not limited thereto.

The agarose is a polysaccharide composed of galactose extracted from seaweed. It is mainly used as a cell culture medium and can be used in the form of hydrogel because the hardness of the structure can be adjusted. Through this, agarose can be seeded evenly inside the scaffold It has advantages. Agarose can also induce gelation by temperature change without ion crosslinking or chemical crosslinking. However, if the agarose solution is out of the above-mentioned content range, gelation may not be performed, or cells may be damaged or killed.

The alginate, collagen and agarose added to the mixture may be added in the form of a storage solution, but are not limited thereto.

The agarose stock solution may be prepared by dissolving agarose in an amount of 1 to 10 parts by weight with respect to 100 parts by weight of distilled water. The agarose stock solution may be prepared by dissolving agarose in an amount of 1 to 3 wt% And the collagen storage solution may be prepared by dissolving collagen in an amount of 20 to 30 parts by weight based on 100 parts by weight of distilled water, but is not limited thereto.

Cells that can be cultured by the three-dimensional cell culture method of the present invention can be selected from normal cells and cancer cells.

The cancer cells may be selected from the group consisting of ovarian cancer, breast cancer, gastric cancer, colorectal cancer, rectal cancer, lung cancer, prostate cancer, kidney cancer, pancreatic cancer, thyroid cancer, uterine cancer, bladder cancer, polymorphic glioblastoma, skin cancer, melanoma, thyroid cancer, May be selected from solid tumor cells consisting of a cell line selected from the group consisting of a biliary tumor, an osteosarcoma, a laryngeal cancer, a oral cancer, a salivary gland cancer, an esophageal cancer, an osteosarcoma, a tongue cancer, a bile duct cancer, a glioma, Such as, but not limited to, blood cancer.

The present invention also relates to a method for producing an alginate solution, Adding a collagen solution to the cultured mixture to prevent cell detachment; Adding an agarose solution to the collagen-containing mixture, allowing the mixture to stand and gel, and culturing the cells three-dimensionally; And analyzing the growth, proliferation, death, differentiation and migration of the three-dimensional cultured cells.

The present invention also relates to a method for producing an alginate solution, Adding a collagen solution to the cultured mixture to prevent cell detachment; Adding an agarose solution to the collagen-containing mixture, allowing the mixture to stand and gel, and culturing the cells three-dimensionally; And analyzing the cell survival rate by adding an anticancer agent to the three-dimensional cultured cells.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

The following reference example is provided to provide a reference example commonly applied to each embodiment according to the present invention.

< Reference Example  1> Cell and cell culture

The types of cell lines used in the following examples are as follows. The mouse T cell lymphoma cell line (EL4) was purchased from Sigma-Aldrich (Saint Louis, Mo., USA), and the human ovarian cancer cell line A2780 cell line expressing chemotherapy- Type Culture Collection (ATCC, Manassas, VA, USA). R182 cells, which are chemotherapy-sensitive human ovarian cancer cell lines, A thymic epithelial cell line (thymic epithelial cell line, TEC), which was provided by J. Shah (Memorial Sloan-Kettering Cancer Center, New York, NY, USA) epithelial cell, CREC) Barna B. Knowles (The Jackson Laboratory, Bar Harbor, ME, USA).

R182 cells and A2780 cell lines were treated with 10% (v / v) fetal bovine serum (FBS; Gibco, Invitrogen), 100 IU / mL penicillin (Gibco, Invitrogen) and 100 ug / mL streptomycin. The cells were cultured and maintained in RPMI-1640 medium (HyClone, Logan, UT, USA) containing 5% CO ₂ at 37 ° C.

The CREC and EL4 cell lines were also stained with 10% (v / v) fetal bovine serum (FBS; Gibco, Invitrogen), 100 IU / mL penicillin (Gibco, Invitrogen) and 100 ug / mL streptomycin. (DMEM, HyClone, Logan, UT, USA) containing Dulbecco's Modified Eagle Medium (Gibco, Invitrogen) at 37 ° C and 5% CO ₂ .

All cell cultures were exchanged for new culture once every 2-3 days.

< Reference Example  2> Statistical analysis

All results were expressed as mean ± SD (SD) for each analysis. Statistical analysis was performed using a two-tailed t-test, and statistical significance was evaluated based on P <0.05.

< Example  1> Agarose -Collagen- Alginate  complex Hydrogel  Support production

A hydrogel support was prepared in the same manner as in Fig.

(Affymetrix, Cleveland, Ohio, USA) was prepared by dissolving sodium alginate (Sigma-Aldrich) in distilled water for 16 hours at a concentration of 3% by weight to prepare a hydrogel support. Was added to sterilized distilled water at a concentration of 2% by weight, and heated to boiling at 100 캜 to prepare an agarose stock solution. In addition, marine biologically derived collagen (hereinafter, referred to as "marine collagen", Geltech, Busan, Korea) was added to distilled water in an amount of 25% by weight and vortexed to prepare a marine collagen storage solution.

Each solution made by the above method was mixed at a temperature of 25 ° C to 37 ° C. First, the cells were mixed with 33.3 μL of a 3 wt% alginate stock solution, mixed with 30 μL or 40 μL of a 25 wt% marine collagen stock solution, and finally added with 6.25 μL of a 2 wt% agarose stock solution. (Hereinafter referred to as &quot; 0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate &quot;, hereinafter referred to as &quot; agarose- Or &quot; 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate &quot;). The mixture was then allowed to stand at 4 ° C for 5-30 minutes, more preferably at 5-10 minutes.

The three-dimensional cell culture using the hydrogel support of the present invention can be carried out by directly mixing the mixture into a culture container to be used for cell culture and gelling the mixture, or when the gel is transferred to another container, The end of the syringe was cut and then the mixture was poured into a gel, and the gel was moved into a 24-well plate using a piston of a syringe to cultivate the cells.

Therefore, when the hydrogel of the present invention is used, a gel can be prepared according to the size and shape of the cell culture container, and the three-dimensional cell culture using the hydrogel support thus prepared is possible.

< Example  2> Ca ^{2 +}  Identification of Cytotoxicity by Cation ( cell cytotoxicity assay )

The cytotoxicity was assessed by a conventional method [HW Lee et al., Regul. Pept. (2008), 147, 72-81]. In the case of the cells of the reference example 1 in 96-well plates containing 10% FBS cell culture medium with 0.1 mL for R182 and A2780 cells with 1 × 10 ⁴ cells / well density, R182 and A2780 cells 1 × 10 ⁵ cells / Cells were plated at well concentration and cultured for 24 hours, followed by further culture for 12-16 hours in the absence of FBS.

The cultured cells were treated with various concentrations of calcium chloride (CaCl ₂ , Sigma-Aldrich) at concentrations of 10, 50, 100, 200 and 300 mM and cultured for 18 to 24 hours. Cell Viability Assay kit, Daeillab Service, Seoul, Korea).

10 μL of WST-1 reagent was added to each well, followed by incubation in a humidified incubator at 37 ° C. and 5% CO ₂ for 2 hours. Then, using a microplate reader (Tecan, M, Switzerland) And absorbance was measured to calculate percent growth rate.

All results were also expressed as mean ± SD (SD) for each analysis. Statistical analysis was performed using a two-tailed t-test, and statistical significance was evaluated based on P <0.05.

As a result, CREC cells were treated with CaCl ₂ (P <0.01), 100.0 to 23.0% (P <0.01), 200 mM to 9.9% (P <0.001) and 300 mM to 10.3% (P <0.01) 0.001) showed a dose-dependent cytotoxic effect, and cytotoxic effects of CaCl ₂ on mouse T cell lymphoma cells (EL4), a kind of cancer cells, were measured. As a result, 10 mM (P <0.001) at 100 mM, 25.2% (P <0.001) at 100 mM, 11.2% (P <0.001) at 200 mM and 9.7% (P < Dependent cytotoxic effect.

In the human ovarian cancer cell, R182 cells, CaCl ₂ (P < 0.001), 25.0% (P < 0.001) at 150 mM, 21.4% (P < 0.001) at 200 mM, and 24.9% P <0.001) and 25.6% (P <0.001) at 300 mM showed a very strong cytotoxic effect at all concentrations above 50 mM. In A2780 cells, which are different types of human ovarian cancer cells, CaCl ₂ (P <0.05) at 200 mM, 39.5% (P <0.05) at 250 mM, and 43.7% at 300 mM, respectively, at 50 mM, 74.9%, 100 mM at 65.9%, 150 mM at 41.5% (P < 0.05), indicating a dose-dependent cytotoxic effect.

From the above results, it was confirmed that CaCl ₂ shows a significant cytotoxic effect on various types of solid cancer and blood cancer cells as well as normal cells at a normal concentration. In this way, CaCl _{2, which} is widely used as a hardener in the gelation process, which is an essential step in the production of an alginate-based hydrogel cell culture supporter which is generally used most as a material of hydrogel-based cell culture supporter, It is very important to develop a method for gelation of hydrogels which can reduce or eliminate such side effects.

< Example  3> Agarose -Collagen- Alginate  complex Hydrogel  Characterization of support material and composition

Agarose alone hydrogel, agarose-alginate complex hydrogel, and agarose-collagen-alginate complex hydrogel were mixed to prepare a composite hydrogel support similar to that of Example 1, Was prepared in the same manner as in Example 1 and the characteristics thereof were analyzed.

The agarose-free hydrogel was prepared at concentrations of 0.5, 1, 1.5, and 2 wt%, which are conventionally used concentrations, and the agarose-alginate complex hydrogel was prepared by mixing 3 wt% alginate stock solution and 2 wt% agarose stock solution And then mixed to finally prepare a composite hydrogel support such that the concentration of alginate was 1 wt% and the concentration of agarose was 0.125 wt%. The agarose-collagen-alginate complex hydrogel was prepared in the same manner as in Example 1.

For cell culture with each hydrogel support prepared by the above method, A2780 cells were treated with 0.5, 1 and 1.5 wt% agarose solely hydrogel and 0.125 wt% agarose-1 wt% alginate complex After adding to the hydrogel support, it was placed in a 96-well plate and gelled at 4 ° C for 5 to 10 minutes. After the culture was carried out, the cells were observed under a phase contrast microscope on days 1, 3 and 5 after culturing. EL4 cells were added to agarose alone hydrogel supports of 0.5, 1, 1.5 and 2 wt%, and then placed in a 96-well plate and gelled at 4 ° C for 5 to 10 minutes. The EL4 cells were cultured in the medium, And on day 10, cell morphology was observed with a phase contrast microscope.

As a result, when the A2780 cells were cultured in agarose alone hydrogel support as shown in FIG. 4, the number of killed cells increased with the lapse of the cell culture period, and all the agarose hydrogels of 0.5, 1 and 1.5 wt% The number of killed cells increased. In the case of the alginate-agarose complex hydrogel support, there was almost no dead cells, but the mechanical strength of the hydrogel was so weak that the cells could not be stably bound to the gel, and the gel was out of the gel.

The EL4 cells were three-dimensionally cultured on hydrogel supports of agarose alone according to various concentrations and incubation times. As a result, referring to FIG. 5, in the case of agarose alone hydrogel supports, the concentration of agarose and the cell culture period , The growth of EL4 cells decreased and the number of apoptotic cells increased. At 0.5% agarose, cell growth slowed at day 2, and at day 5, there was little growth and the number of dead cells increased. In 1 wt% agarose, cell growth was slower than 0.5 wt% agarose, and the number of dead cells was more confirmed. The 1.5 wt% agarose also showed a decrease in cell growth and the number of dead cells . Particularly, in the 2 wt% agarose, almost no growth of cells was observed from the second day after the incubation, and all the cells were observed to be killed over time.

On the other hand, when the animal collagen was added to the agarose-collagen-alginate complex hydrogel support prepared according to the present invention so that the final concentration was 0.05 wt% in the mixed solution as shown in Figs. 6 and 7, about 4,500 EL4 cells The final concentration was 230 to 230, the final concentration was 0.1 to 20, 20500, the final concentration was 0.2 to 18, 000, the final concentration was 0.5 to 17500 at the final concentration, From 1 wt% to 11,500, the final concentration from 2 wt% to 5,500, and the final concentration from 5 wt% to 4,000 desorbed cells.

From the above results, the survival and growth of A2780 and EL4 cells were excellent in the agarose-alginate complex hydrogel support as compared with the hydrogel support of agarose alone, and the agarose-agarose complex hydrogel support was superior to the agarose- And exhibited stable cell binding ability in the hydrogel support.

Therefore, the cell culture using the agarose-collagen-alginate complex hydrogel support is superior to the agarose alone hydrogel in cell survival and growth, and solves the cell desorption phenomenon of the agarose-alginate complex hydrogel support having weak mechanical strength , And it was found to be very suitable for three-dimensional cell culture.

< Example  4> Animal collagen  Or marine collagen use

As described above, in the case of the three-dimensional cell culture method using the agarose-alginate complex hydrogel support, it was confirmed that the cells were desorbed out of the gel. However, it has been confirmed that the problem of cell desorption is solved through a three-dimensional cell culture method using an agarose-collagen-alginate complex hydrogel support to which collagen has been added. Therefore, in order to confirm the efficiency of collagen, animal collagen and marine collagen .

(0.05, 0.1, 0.2, 0.5, 1, 2, 5 and 10) weight of 0.125 wt% agarose-0.05 wt% rat tail collagen-1.0 wt% alginate and 0.125 wt% agarose % Marine collagen-1.0 wt% alginate composite hydrogel support was prepared, and EL4 cells of 5x10 ⁴ cells / gel were included in the preparation of each of the hydrogel supports. Then, 80 μL of the mixed solution was added to a 1 mL syringe, and the mixture was gelled at 4 ° C. for 5 to 10 minutes. The mixture was then placed in a 48-well plate and cultured as shown in FIG. 8. The cells were observed on day 1, day 2, day 3, Respectively.

(0.05, 0.1, 1, 3, 5, 7.5, 10 and 15) weight% of marine collagen-1.0 weight% agarose-0.05 weight% of rat tail collagen -1.0 weight% of alginate and 0.125 weight% Alginate complex hydrogel supports were prepared, and A2780 cells were included in the preparation of each of these hydrogel supports. Then, the mixed solution was put into a 96-well plate and gelled at 4 ° C for 5 to 10 minutes, and then cultured in the medium. Cells were observed under a phase contrast microscope on days 1 and 3 after culturing as shown in FIG.

As a result, as shown in FIG. 9 in which EL4 cells were observed and FIG. 8 in which A2780 cells were observed, it was found that two types of agarose-collagen-alginate complex hydrogel supports using 0.05 wt.% Animal collagen and 5-10 wt.% Marine collagen The cell growth of the agarose-collagen-alginate complex hydrogel support showed the same effect. However, in the case of marine collagen, marine collagen should be used at a concentration about 100 times higher than that of animal collagen in order to exhibit the same effect as animal collagen. However, since marine collagen is about 10,000 ~ 100,000 times cheaper than animal collagen, It was confirmed that the agarose-collagen-alginate complex hydrogel support exhibited similar effects to the complex hydrogel support using animal collagen and could be used economically very usefully.

< Example  5> Agarose - Marine collagen - Alginate  complex Hydrogel  Identify the transparency properties of the support

In order to confirm the transparency of the agarose-marine collagen-alginate complex hydrogel support, 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel support prepared in the same manner as in Example 1, ⁴ × 10 ⁴ cells / gel of EL4 cells were added to the nanofiber support prepared by spinning, the hydrogel support formed by the chemical crosslinking method by 100 mM CaCl ₂ , and the plate for the conventional two-dimensional 96-well cell culture After the incubation, the transparency of each support was compared through observation of phase contrast microscope.

 As a result, as shown in Fig. 10, unlike the hydrogel support (b) formed by a commonly used ionic chemical crosslinking method and the nanofiber support (c) produced by electrospinning, which is widely used in tissue engineering, As can be seen from the two-dimensional cell culture (a), since the agarose-marine collagen-alginate complex hydrogel support (d) of the present invention provides very high transparency, it is easy to observe the morphology of EL4 cells using a phase- . Therefore, the agarose-marine collagen-alginate complex hydrogel support of the present invention can be used to observe various types of cells that can not be observed in an alginate-based hydrogel or a nanofiber-based support crosslinked with ions such as calcium in a three-dimensional cell culture using a phase contrast microscope .

< Example  6> Agarose - Marine collagen - Alginate  complex Hydrogel  Analysis of Physical Properties of Support

One. Hydrogel  Support production

As a test group used for analyzing the structure of the support, first, 1% by weight of alginate alone hydrogel, 0.125% by weight of agarose alone hydrogel, 3) 7.5% by weight of marine collagen alone hydrogel, Agarose-1 wt.% Alginate complex hydrogel, sixth, 0.125 wt.% Agarose-7.5 wt.% Marine collagen-1 wt.% Alginate complex hydrogel seventh, 0.125 wt.% Agarose-10 Wt% marine collagen-1 wt% alginate complex hydrogel was prepared in the same manner as in Example 1.

2. Using a scanning electron microscope Hydrogel  Analysis of support structure

The hydrogel supports of the 7 experimental groups prepared in Example 6-1 were dispensed in a 1 mL syringe at a rate of 100 μL, and gelled at 4 ° C. for 5 to 10 minutes. The cells were frozen at -20 ° C. for 24 hours, (FDS series, ilShinBioBase) for 24 hours. The surface was treated with an ion sputter (E-1010, Hitachi, Japan) and observed under a scanning electron microscope (JSM-6490LV, JEOL, Japan) under an accelerating voltage of 20 kV.

As a result, when cross sections of 1 wt% alginate, 0.125 wt% agarose, 7.5 and 10 wt% marine collagen solely hydrogel, and 0.125 wt% agarose-1 wt% alginate complex hydrogel support were observed, Although it was composed of an irregular pore structure as a whole, it was observed that the cross section of the 0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate or 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel support , It can be seen that the structure of the pore is regularly formed as a whole.

3. FT - IR  ( Fourier transform infrared spectroscopy ) analysis

The hydrogel supports of the 7 experimental groups prepared in Example 6-1 were dispensed in a 1 mL syringe at a rate of 100 μL, and gelled at 4 ° C. for 5 to 10 minutes. The cells were frozen at -20 ° C. for 24 hours, (FDS series, ilShinBioBase) for 24 hours and measured by an infrared spectroscopy system (FT-IR Microscopy System).

As a result, as shown in FIG. 12, the FT-IR spectrum of the 0.125% by weight agarose-1% by weight alginate complex hydrogel support showed the property of the parent molecule, and no significant additional band was present. The FT-IR spectrum of the 0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate complex hydrogel support showed the properties of a parent molecule, particularly 7.5 wt% marine collagen, Was not present.

From the above results, it was confirmed that the main component of FT-IR spectrum of 0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate complex hydrogel support was mainly 7.5 wt% marine collagen.

Similarly, the FT-IR spectrum of the 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel support complex hydrogel support showed 10 wt% marine collagen character as a parent molecule , There were no significant additional bands. This FT-IR spectrum of the 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel support shows mainly 10 wt% marine collagen as its main component.

From the above results, no new chemical cross-links were formed in the agarose-marine collagen-alginate complex hydrogel support, and chemical cross-links were observed in the agarose-marine collagen-alginate complex hydrogel support. It is confirmed that the gelation occurs due to physical bonding such as hydrogen bonding or other interaction depending on the physical properties of the constituent molecules.

This is because a hydrogen bond is formed between a proton donor and a proton acceptor present in a molecule. As shown in FIG. 13, an interaction between an ocean collagen and an agarose and an ocean collagen and an alginate is hydrogen bonding hydrogen bonds, OH, -NH ₂ , and C═O groups present in the collagen can form hydrogen bonds with the OH groups present in the agarose and OH and C═O present in the alginate It is because.

4. Measurement of swelling behavior (water content; swelling kinetics )

The hydrogel supports of the seven experimental groups prepared in Example 6-1 were dispensed in a 1 mL syringe in a volume of 100 μL, gelled at 4 ° C for 5-10 minutes, and dried at 40 ° C for 24 hours. The dried agarose-marine collagen-alginate complex hydrogel support was weighed, immersed in phosphate buffered saline (PBS) and distilled water at 37 ° C., Respectively. The weighing was carried out at room temperature, and the solution flowing down was removed and measured. The water content was calculated by the following formula.

Water content (%) = [(weight of swollen gel - weight of dried gel) / weight of swollen gel] × 100

As a result, as shown in Fig. 14, 0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate complex hydrogel, and 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate composite hydrogel support Was slightly delayed compared to the distilled water. This is probably due to osmotic pressure of agarose-marine collagen-alginate complex hydrogel support in distilled water rather than PBS.

In addition, these supports exhibited fairly rapid swelling and reached an equilibrium state within 2 hours.

< Example  7> Agarose - Marine collagen - Alginate  complex Hydrogel  Analysis of three-dimensional culture characteristics of cells in scaffolds

 One. Hydrogel  Three-dimensional cell culture using cell culture supporter

0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel support was prepared in the same manner as in Example 1, cells were added, and the cells were placed in a 96-well plate and incubated at 4 ° C for 5-10 min Lt; RTI ID = 0.0 &gt; g. &Lt; / RTI &gt;

2. Actin staining  And hematoxylin-eosin staining Spheroidal  Formability analysis

 To perform F-actin staining in whole gel of human ovarian cancer cell A2780 cultured three-dimensionally for 7 days using agarose-marine collagen-alginate complex hydrogel support, the cell-cultured complex hydrogel support was washed twice with PBS After washing, the cells were fixed with 100% methanol fix solution at -20 ° C for 20 minutes. Then, the cells were washed twice with PBS, followed by permeation to 0.1% triton-x 100 solution at room temperature for 5 minutes, followed by washing twice with PBS. To remove nonspecific staining, the cells were treated with 1% bovine serum albumin (BSA) solution at room temperature for 1 hour and then washed twice with PBS. Then, actin-detecting Phalloidin-FITC (Promega, Madison, Wis., USA) was diluted 1: 50 in 0.2% BSA solution and reacted at 4 ° C for 18 hours. Then, tris-buffered saline (TBS), and the complex hydrogel support was placed on a cover glass. The cells were stained with DAPI (Vector Lab, Inc., Burlingame, CA, USA) , And analyzed by fluorescence microscopy.

Hematoxylin-eosin (H-E) staining was also performed on human ovarian cancer cells (A2780) cultured three-dimensionally for 7 days using an agarose-marine collagen-alginate complex hydrogel support. First, the complexed hydrogel support on which the cells were cultured was washed once with PBS and fixed with 100% methanol fixative at 20 DEG C for 20 minutes. The complex hydrogel supports were nuclear stained with hematoxylin for 5-10 minutes, washed with water, and then stained cytoplasm for 5-7 minutes in eosin. After washing with 100% ethanol, a part of the composite hydrogel support was peeled off, placed on a slide, covered with cover glass, and observed with an optical microscope.

As a result, when the results of the optical microscope (a), hematoxylin-eosin staining (b), DAPI (c) and F-actin staining (d) . &Lt; / RTI &gt; Therefore, it has been confirmed that the three-dimensional cell culture method using the agarose-marine collagen-alginate complex hydrogel support induces highly effective cell spoloids.

As shown in FIG. 16, after EL4 cells were cultured for 1, 3, and 5 days, they were observed with a phase contrast microscope. As a result, it was confirmed that the formation of the cell spoloid was very regular and the cell spoiloid was effectively induced as the incubation period was increased.

From the above results, it was confirmed that the three-dimensional cell culture method using the agarose-marine collagen-alginate complex hydrogel support can be very useful for studying the mechanism of interaction with the microenvironment of cancer cells.

3. Speroid Burding ( spheroid budding ) Formability  analysis

In order to analyze the spheroid budding phenomenon which plays an important role in the formation of the cell spoloids in the previous experiment, A2780 and R182 cells were cultured in agarose-marine collagen-alginate complex hydrogel support for 7 days, The gel was subjected to hematoxylin-eosin staining and observed under an optical microscope. At the same time, unstained cells were observed with a phase contrast microscope.

As a result of analyzing the result of phase contrast microscope (a) and hematoxylin-eosin staining (b) as shown in Fig. 17, spheroid budding phenomenon was very effectively confirmed. Therefore, it has been confirmed that the three-dimensional cell culture method using the agarose-marine collagen-alginate complex hydrogel support can be effectively used for studying the spheroid budding phenomenon.

4. Within spalloid Drug diffusion ability  analysis

In order to evaluate the diffusion ability of a drug reaching cancer cells in a three-dimensional environment using an Agarose-marine collagen-alginate complex hydrogel support, a solution of human ovarian cancer cell (A2780) using an agarose-marine collagen-alginate complex hydrogel support The degree of drug diffusion was measured using a three - dimensional culture model.

First, the cells were cultured until the sprooid was formed in the agarose-marine collagen-alginate complex hydrogel support, and then the anticancer drug doxorubicin (Cell Signaling Technology, Danvers, MA, USA) Respectively. After hydrogel supports were washed with PBS, they were nuclear stained with DAPI solution and the distribution of doxorubicin in the spheroids was confirmed by confocal laser microscopy (FV1000-IX81, Olympus, Japan).

As a result, it was confirmed that doxorubicin reaches cancer cells in all regions within the sphere as shown in Fig.

From the above results, it was confirmed that the three-dimensional cancer cell culture model using the agarose-marine collagen-alginate complex hydrogel support can be very usefully used for the screening method for the sensitivity test of various drugs including anticancer drugs.

< Example  8> Agarose - Marine collagen - Alginate  complex Hydrogel  Cells through three-dimensional cell culture using supporter Growth capacity  analysis

One. Hydrogel  Three-dimensional cell culture using cell culture supporter

Agarose-7.5 wt% marine collagen and 1 wt% alginate complex hydrogel support were prepared in the same manner as in Example 1, and A2780 cells were added thereto. The cells were then added to a 96-well plate, Min, and then cultured in the medium. In addition, conventional two-dimensional cell culture was also performed as a control for three-dimensional cell culture.

2. Cell growth ability  Measure( cell growth assay )

A2780 cells as human ovarian cancer cells at ⁵ × 10 ⁵ cells / gel were cultured on agarose-marine collagen-alginate complex hydrogel support, and the generation of spheroids of cells was observed with a phase contrast microscope after 0, 1, 3, 5 and 7 days . Spheroid sizes were measured on days 5 and 7. In addition, the WST-1 reagent was treated for 2 hours and the absorbance at 450 nm was measured using a microplate reader to calculate the percentage growth rate.

As a result, as shown in FIGS. 19A and 19B, the cells proliferated more rapidly in the two-dimensional environment at the initial stage of culture, but they proliferated more rapidly in the three-dimensional environment after five days of culture. In addition, as shown in FIG. 19C, it was confirmed that the size of the cell spoiloid was further increased in the three-dimensional environment at the 7th day group than at the 5th day group.

From the above results, it was confirmed that the three-dimensional cell culture method using the agarose-marine collagen-alginate complex hydrogel support is more suitable for the formation of a three-dimensional cell culture environment than a two-dimensional cell culture, It was confirmed that the cell supports can be used to construct suitable models for cell culture studies.

3. CFSE  Dye analysis

CFSE staining was performed to confirm cell proliferating ability in an agarose-marine collagen-alginate complex hydrogel support. EL4 and CREC cells were treated with CellTracker ^TM Green CMFDA (Invitrogen, Carlsbad, Calif., USA) at 37 ° C for 8 minutes and then washed twice with PBS. Cells labeled with CFSE were cultured on a complex hydrogel support for 0, 1, 2, and 3 days, and cell proliferative activity was observed using a fluorescence microscope.

As a result, as shown in FIG. 20, it was confirmed that the cells cultured in the agarose-marine collagen-alginate complex hydrogel support decreased in number of CFSE-positive cells over time. Therefore, it was confirmed that when cultured in the agarose-marine collagen-alginate complex hydrogel support of the present invention, the proliferation of EL4 cells was induced.

In addition, when EL4 and CREC cells were co-cultured, EL4 cell proliferation was slower than EL4 cell culture alone.

From the above results, it was confirmed that the agarose-marine collagen-alginate complex hydrogel support of the present invention not only provides a three-dimensional environment suitable for proliferation of CREC and EL4 cells, but also can be used as a very useful means for studying cell proliferation Respectively.

< Example  9> Agarose - Marine collagen - Alginate  complex Hydrogel  Cells through three-dimensional culture of cells in supporters Survival  analysis

One. Hydrogel  Three-dimensional cell culture using cell culture supporter

0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate complex hydrogel support was prepared in the same manner as in Example 1, cells were added, and the cells were added to a 96-well plate and incubated at 4 ° C for 5-10 min Lt; RTI ID = 0.0 &gt; g. &Lt; / RTI &gt;

2. Live / Dead  Dye analysis

In order to confirm whether or not the hydrogel support of the present invention shows cytotoxicity in cell culture using the agarose-marine collagen-alginate complex hydrogel support of the present invention, agarose prepared by the method of Example 8-1 Human ovarian cancer cells (A2780) were cultured in marine collagen-alginate complex hydrogel supports for 7 days and 10 days, and EL4 and CREC cells were cultured alone or co-cultured for 2 weeks. After washing the complex hydrogel support with PBS once, 2 μL calcein AM and 0.5 μL EthD-1 (LIVE / DEAD Viability Kit, Molecular Probes) were diluted in 1 mL PBS, stained in the dark for 30 minutes, And analyzed with a microscope (FV1000-IX81, Olympus, Japan).

As a result, the survival rate of human ovarian cancer cells cultured in Agarose-marine collagen-alginate complex hydrogel support was very high as shown in FIG. Also, referring to Figure 22, the survival rates of EL4 and CREC cells cultured in agarose-marine collagen-alginate complex hydrogel supports were very high.

From the above results, it was confirmed that the agarose-marine collagen-alginate complex hydrogel support of the present invention does not show cytotoxicity.

< Example  10> Agarose - Marine collagen - Alginate  complex Hydrogel  Three-dimensional cancer cell culture in supporter Malignancy  analysis

One. Hydrogel  Three-dimensional cell culture using cell culture supporter

In the case of EL4 cells, 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel support was prepared in the same manner as in Example 1, and cells were added and the mixture was added to a 1 mL syringe with 80 μL And the mixture was gelled at 4 ° C for 5-10 minutes, and then cultured in a 48-well plate. In the case of A2780 cells, 0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate complex hydrogel support was prepared in the same manner as in Example 1, cells were added, and the cells were added to 96- For 5-10 minutes, and then cultured in the medium. In addition, conventional two-dimensional cell culture was also performed as a control for three-dimensional cell culture.

2. Semi-quantitative Reverse transcription  Polymerase chain reaction analysis ( semiquantitative reverse transcription - 중합체  chain reaction 분석 , RT - PCR )

EL4 cells were cultured in two- and three-dimensional environments in an agarose-marine collagen-alginate complex hydrogel support, and the degree of expression of notch closely related to the malignancy of cancer cells was confirmed by RT-PCR analysis.

EL4 cells were cultured in a complex agarose-marine collagen-alginate hydrogel support for 2 weeks in order to observe the expression of Notch by reverse transcription-polymerase chain reaction (hereinafter "RT-PCR"

After that, 1.5 mL of QG buffer (Qiagen, Valencia, CA, USA) and 2 mL of RLT buffer (Qiagen) were mixed with a complex agarose-marine collagen-alginate hydrogel support and incubated at room temperature with a complex agarose-marine collagen-alginate hydrogel After dissolving the support, Total RNA was isolated from cultured cells according to the manufacturer's instructions using trizol reagent (Ambion, Invitrogen).

(DT) 12-18 oligo (dT) 12-18] Oligo (dT) primer, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ , 40 mM DTT, 0.5 mM deoxynucleotide triphosphate (dNTP) mixture, 10 units RNase inhibitor and 200 units Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) was used to synthesize single-stranded cDNA from 2 g of total RNA.

PCR reactions for mouse Notch-1, Notch-2 and GAPDH gene amplification were performed using mouse Notch-1 forward and reverse primers of SEQ ID NOs: 1 and 2 in Table 1, mouse Notch-1 and SEQ ID NO: -2 omni-directional and reverse primers, or 0.4 pmol of mouse GAPDH forward and reverse primers of SEQ ID NOs: 5 and 6 and 1 μL cDNA sample, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl ₂ , 0.1 Initial denaturation was carried out at 94 ° C for 3 minutes using 25 μl of a buffer solution containing 5% Triton X-100, 0.2 mM dNTP and 5 unit Taq DNA polymerase (Promega) 30 sec., 60 sec., 30 sec. And 72 sec., 30 sec. Reaction was performed in 35 cycles. In the case of GAPDH, the reaction was carried out in 30 cycles.

The PCR reactions for human snail, vimentin, CD44 and GAPDH gene amplification consisted of the human snail forward and reverse primers of SEQ ID NOS: 7 and 8, the reverse primer of SEQ ID NO: And 10 human vimentin forward and reverse primers, human CD44 forward and reverse primers of SEQ ID NOs: 11 and 12, human endothelin-1 forward and reverse primers of SEQ ID NOs: 13 and 14, Or 0.4 pmol of the human GAPDH forward and reverse primers of SEQ ID NOS: 15 and 16 and 1 μL cDNA sample, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl ₂ , 0.1% Triton The initial denaturation of CD44, Snail and GAPDH was performed at 94 ° C for 5 minutes using 25 μL of buffer solution containing X-100, 0.2 mM dNTP and 5 unit Taq DNA polymerase (Promega) , 30 cycles of reaction at 94 DEG C for 30 seconds, reaction at 57 DEG C for 30 seconds and reaction at 72 DEG C for 30 seconds in 27 cycles It was.

In the case of vimentin, initial denaturation was carried out at 94 ° C for 5 minutes, followed by 27 cycles at 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. Finally, endothelin-1 was subjected to initial denaturation at 94 ° C for 5 minutes, followed by initial denaturation at 94 ° C for 30 seconds, at 65 ° C for 30 seconds, and at 72 ° C for 30 seconds for 27 cycles ).

After the PCR reaction, the reaction product samples produced by 2% agarose gel electrophoresis were stained with EtBr (Ethidium bromide) and analyzed under UV light illumination.

As a result, the expression of both Notch-1 and Notch-2 was increased in the lymphoma cells cultured in the three-dimensional environment rather than the two-dimensional culture as shown in Fig. This is the result of confirming that the cancer cells cultured in the three - dimensional environment are increased in malignancy unlike the two - dimensional environment.

From the above results, it was confirmed that the three-dimensional cancer cell culture model using the agarose-marine collagen-alginate complex hydrogel support according to the present invention can effectively simulate a living body (in-vivo) environment.

In addition, in the human ovarian cancer cell host A2780 cells cultured in a two-dimensional and three-dimensional environment, the malignancy of the cancer cells, particularly, the snail and the bee, which are closely related to the epithelial-mesenchymal transition (EMT) CD19, an important marker for vimentin, cancer stem cells, and endothelin-1, which plays a key role in tumor growth and metastasis.

As a result, it was confirmed that the cancer cells cultured in the three-dimensional environment more than the two-dimensional environment as shown in Fig. 24 increase the expression of the marker related to the cancer malignancy.

From the above results, it was confirmed that the culture conditions of cancer cells such as growth, progression, metastasis and malignancy of cancer are very similar to those of living body, in the three-dimensional cancer cell culture based on agarose-marine collagen-alginate complex hydrogel support. Therefore, the three-dimensional cell culture method using the agarose-marine collagen-alginate complex hydrogel support of the present invention is useful as an in-vivo biomarker environment that can not be provided by the conventional two-dimensional cell culture method, , Can be usefully used for the development of therapeutic agents for cancer cells, as well as research on growth, metastasis and malignancy.

gene SEQ ID NO: Primer sequence mouse Notch-1
One (Forward) 5'-CCCAGCAGGTGCAGCCACAG-3 ' 2 (Reverse direction) 5'-GGTGATCTGGGACGGCATGG-3 ' mouse Notch-2
3 (Forward) 5'-ATGTGGACGAGTGTCTGTTGC-3 ' 4 (Reverse direction) 5'-GGAAGCATAGGCACAGTCATC-3 ' mouse GAPDH
5 (Forward) 5'-GAAATCCCATCACCATCTTCCAGG-3 ' 6 (Reverse) 5'-GAGCCCCAGCCTTCTCCATG-3 ' Human Snail
7 (Forward) 5 '-AGACCCACTCAGATGTCAA-3' 8 (Reverse) 5'-CATAGTTAGTCACACCTCGT-3 ' Human vimentin
9 (Forward) 5'-GAGGAGATGCGGGAGCTGCG -3 ' 10 (Reverse) 5'-CTGCCCTGCGTGACGTACGT-3 ' Human CD44 11 (Forward) 5` -CTCCAGTGAAAGGAGCAGCA-3` 12 (Reverse direction) 5` -AGCAGGGATTCTGTCTGTGC-3` Human endothelin-1
13 (Forward) 5`-TGCTCCTGCTCTTCCCTGATGGATAAAGAGTGTGTC-3` 14 (Reverse direction) 5`-GGTCACATAACGCTCTCTGGAGGGCTT-3` Human GAPDH
15 (Forward) 5'-TGGAGAAACCTGCCAAGTATG -3 ' 16 (Reverse direction) 5'-TTGTCATACCAGGAAATGAGC-3 '

< Example  11> Agarose - Marine collagen - Alginate  complex Hydrogel  Measurement of Anticancer Drug Susceptibility for Three-Dimensional Cultured Cancer Cells in Support: Analysis of Suitability as an In-Bibosomal Anticancer Susceptibility Measurement Model

One. Hydrogel  Three-dimensional cell culture using cell culture supporter

In the case of EL4 cells, 0.125 wt% agarose-10 wt% marine collagen-1 wt% alginate complex hydrogel support was prepared in the same manner as in Example 1, and cells were added and the mixture was added to a 1 mL syringe with 80 μL And the mixture was gelled at 4 ° C for 5-10 minutes, and then cultured in a 48-well plate. In the case of A2780 cells, 0.125 wt% agarose-7.5 wt% marine collagen-1 wt% alginate complex hydrogel support was prepared in the same manner as in Example 1, cells were added, and the cells were added to 96- For 5-10 minutes, and then cultured in the medium. In addition, conventional two-dimensional cell culture was also performed as a control for three-dimensional cell culture.

2. Cell growth ability  Measure( cell growth assay )

In order to conduct a sensitivity test on an anticancer agent, EL4 and human ovarian cancer cell A2780 were cultured in a two-dimensional and three-dimensional environment in an agarose-marine collagen-alginate complex hydrogel support, and the efficacy of the anticancer agent was examined.

EL4 cells cultured in two-dimensional culture conditions and EL4 cells cultured three-dimensionally on an agarose-marine collagen-alginate complex hydrogel support were treated with 5 μM doxorubicin (5927, cell signaling) and 100 μM resveratrol For the human ovarian cancer cell A2780, paclitaxel was treated at a concentration of 0.5 μM for 24 hours, treated with WST-1 reagent, and absorbance was measured at 450 nm using a microplate reader to calculate percent growth rate.

In addition, A2780 cells cultured three-dimensionally for 7 days in A2780 and agarose-marine collagen-alginate complex hydrogel supports cultured under 2-dimensional culture conditions were treated with paclitaxel 10 and 20 μM, curcumin 30 and 50 μM 2 DMSO at the same concentration as the experimental group was administered to the control group. The WST-1 reagent was then treated and the absorbance at 450 nm was measured using a microplate reader to calculate the percentage growth rate.

As a result, as shown in FIG. 25A, when 5 μM of doxorubicin was administered to EL4 cells as a mouse lymphoma cell, the survival rate was 58.6% (P <0.001) in the two-dimensional environment as compared with the control, Cell survival rate was 71.4% (P <0.01) compared to the control group. As shown in FIG. 25B, when resveratrol was treated at a concentration of 100 μM, the survival rate was 50.2% (P <0.001) in the two-dimensional environment as compared with the control, and 93.5% Cell viability.

When paclitaxel was treated with 0.5 μM of human ovarian cancer cell A2780 as shown in FIG. 25C, the survival rate was 60.9% (P <0.001) in the two-dimensional environment as compared with the control group. In the three-dimensional environment, And the cell viability was 94.2%.

Based on the above results, it was confirmed that the three-dimensional cancer cell culture model using the agarose-marine collagen-alginate complex hydrogel support according to the present invention can be very useful for studying the drug resistance mechanism against the anticancer agent and the sensitivity to the anticancer agent.

In addition, A2780 cells were cultured in a two-dimensional and three-dimensional environment in an agarose-marine collagen-alginate complex hydrogel support, and paclitaxel and curcumin efficacy tests for ovarian cancer were conducted. As a result, The survival rate was 12.3% (P <0.01) in the case of paclitaxel treated with 10 μM and 6.4% (P <0.001) in the case of 20 μM in the two-dimensional environment. The survival rate of paclitaxel treated with 10 μM was 40.2% (P <0.001) and the survival rate was 51.4% (P <0.05) at 20 μM.

When curcumin was treated at a concentration of 30 and 50 μM, cell viability was 42.5% (P <0.001) for 30 μM and 36.7% (P <0.001) for 50 μM in the two- And the cell viability was 30.0 μM in the 3 - D environment and 82.4% in the 50 μM cell.

From the above results, it was confirmed that the drug resistance to the anticancer drug effectively appeared in the three-dimensional environment rather than the two-dimensional environment. Through this, it was confirmed that the three - dimensional cancer cell culture model using agarose - marine collagen - alginate complex hydrogel support was excellent as a drug resistance mechanism study for anticancer drugs and a method for testing susceptibility to anticancer drugs.

3. Morphological characteristics of cells and Cytokaratin ( cytokeratin ) dyeing

Human ovarian cancer cells were cultured in a three-dimensional environment in an agarose-marine collagen-alginate complex hydrogel support, and immunostaining was performed with an antibody against cytokeratin, which is a marker specifically expressed in epithelial cells.

First, paclitaxel, an anticancer drug known to have a high killing effect on human ovarian cancer cells in a conventional two-dimensional culture after three-dimensional culture of A2780 cells on the agarose-marine collagen-alginate complex hydrogel support of the present invention, 10 μM and curcumin 30 μM for 2 days, respectively. Control group was treated with DMSO at the same concentration as the experimental group for 2 days. The agarose-marine collagen-alginate complex hydrogel support was then washed once with PBS and fixed in a 100% methanol solution at 20 ° C for 20 minutes. To remove nonspecific staining, the cells were treated with 1% BSA solution for 1 hour at room temperature and stained with plate-cytokaryatin antibody (Sigma-Aldrich) diluted 1:50 for 18 hours at 4 ° C . After washing once with PBS, the cells were stained with DAPI solution for 30 minutes at room temperature and observed with a fluorescence microscope. At the same time, unstained cells were observed with a phase contrast microscope.

As a result, unlike the two-dimensional environment shown in FIG. 27, the cells cultured in the three-dimensional environment effectively showed the drug resistance to the anticancer drug.

From the above results, it was confirmed that the three-dimensional cancer cell culture model using the agarose-marine collagen-alginate complex hydrogel support according to the present invention can be very useful for studying the drug resistance mechanism against the anticancer agent and the sensitivity to the anticancer agent.

< Example  12> Agarose - Marine collagen - Alginate  complex Hydrogel  For cancer cells cultured three-dimensionally in supporter Cancer stem cell Formability  And cancer cells Ability to distinguish  Measurement: In-Bibo Amniotic stem cell induction / Analysis of fitness as growth and cancer cell differentiation model

One. Hydrogel  Three-dimensional cell culture using cell culture supporter

0.125% agarose-10% by weight marine collagen-1% by weight alginate complex hydrogel support was prepared at the final concentration in the same manner as in Example 1, and EL4 cells were added, and 80 μL of the mixed solution was added to a 1 mL syringe Gelled at 4 ° C for 5-10 minutes, and then placed in a 48-well plate and cultured.

2. Flow cell  Analyzer ( flow cytometer Analysis of cell surface molecule expression using

EL4 cells were cultured for 10 days in a three-dimensional (3D) agarose-marine collagen-alginate complex hydrogel support and a 96-well plate in a two-dimensional environment (2D), and then analyzed using a flow cytometer (FACS canto Ⅱ, flow cytometer BD Biosciences).

A 40 [mu] m cell strainer (SPL Life Sciences) was used to separate the cells cultured in the hydrogel support, and 2D cells were isolated using conventional methods. The separated cells were washed twice with Hank's Balanced Salt Solution (HBSS, GibcoLife Technologies) and resuspended in 3 mL of FACS buffer (0.1% BSA, 0.1% sodium azide, HBSS 500 mL) Followed by centrifugation. After the supernatant was removed, the cells were resuspended and the cells were pre-incubated with anti-FcγRII / III (2.4G2, hybridoma supernatant) to avoid nonspecific binding with the antibody and then incubated with HBSS containing 2% FBS (FITC-CD4 or PE-CD4) conjugated with FITC or PE, an APC-conjugated anti-CD8 antibody, a primary anti-mouse monoclonal antibody, (PE-CY7-c-kit) and APC-conjugated anti-sca-1 antibody (APC-sca-1) mu] g / mL for 30 minutes under ice cooling.

Background fluorescence was determined through cells that were reacted with isotype-matched nonreactive antibodies.

The cells were then washed twice with HBSS, resuspended in 500 μL of FACS buffer, and analyzed for expression of cell surface molecules using a flow cytometer using the Dot-plot method. The obtained results were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

As a result, the proportion of c-kit ⁺ sca-1 ⁺ cells corresponding to cancer stem cells among the EL4 cells cultured for 10 days under the 2D environment was 4.6% The proportion of c-kit ⁺ sca-1 ⁺ c-kit cells in one EL4 cell was greatly increased to 31.9%. This is the result of confirming that the culture environment of cancer cells such as the formation and growth of cancer stem cells is very similar to that of living body in the three-dimensional cancer cell culture based on agarose-marine collagen-alginate complex hydrogel support.

Therefore, the three-dimensional cell culture method using the agarose-marine collagen-alginate complex hydrogel support according to the present invention provides an in-vivo culture environment that could not be provided by the conventional two-dimensional cell culture method, It can be used for the study of the anticancer drug resistance mechanism and the development of high efficiency chemotherapy.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Pusan National University Industry-University Cooperation Foundation <120> Three dimensional cell culture method using agarose, collagen and          alginate hydrogel composite scaffold <130> ADP-2014-0164 <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cccagcaggt gcagccacag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggtgatctgg gacggcatgg 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atgtggacga gtgtctgttg c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggaagcatag gcacagtcat c 21 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gaaatcccat caccatcttc cagg 24 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gagccccagc cttctccatg 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 agacccactc agatgtcaa 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 catagttagt cacacctcgt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gaggagatgc gggagctgcg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ctgccctgcg tgacgtacgt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctccagtgaa aggagcagca 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 agcagggatt ctgtctgtgc 20 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tgctcctgct cttccctgat ggataaagag tgtgtc 36 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 ggtcacataa cgctctctgg agggctt 27 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 tggagaaacc tgccaagtat g 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ttgtcatacc aggaaatgag c 21

Claims (11)

Mixing and culturing the alginate solution and the cells;
Adding a collagen solution to the cultured mixture to prevent cell detachment; And
Adding the agarose solution to the collagen-added mixture, and allowing the mixture to stand and gel. The method according to claim 1,
Incubating the alginate solution and the cells at a temperature of 25 to 37 DEG C for 5 to 30 minutes;
Adding a collagen solution to the cultured mixture to prevent desorption of the cells at a temperature of 25 to 37 DEG C for 5 to 30 minutes; And
Adding the agarose solution to the collagen-added mixture, and allowing the gelatin to stand for 5 to 30 minutes at a temperature of 4 to 25 ° C. The three-dimensional cell culture method according to claim 1 or 2, wherein the collagen is marine-derived collagen. The 3-dimensional cell culture method according to claim 1 or 2, wherein 0.5 to 2 parts by weight of alginate is added to 100 parts by weight of the cultured mixture. The 3-dimensional cell culture method according to claim 1 or 2, wherein 1 to 30 parts by weight of collagen is added to 100 parts by weight of the mixture. The method according to claim 1 or 2, wherein agarose is added in an amount of 0.1 to 0.5 parts by weight based on 100 parts by weight of the mixture. The method according to claim 1 or 2, wherein the cells are selected from the group consisting of normal cells and cancer cells. 8. The method of claim 7, wherein the cancer cells are selected from the group consisting of ovarian cancer, breast cancer, gastric cancer, colon cancer, rectal cancer, lung cancer, liver cancer, prostate cancer, renal cancer, pancreatic cancer, thyroid cancer, uterine cancer, Wherein the solid tumor cell is a solid cancer cell selected from the group consisting of laryngeal cancer, hydrocephalus, pyelonephrosis, guinea, salivary gland cancer, esophageal cancer, osteosarcoma, tongue cancer, biliary cancer, glioma, bone cancer and unknown cause cancer. [Claim 7] The 3-dimensional cell culture method according to claim 7, wherein the cell is a blood cancer cell selected from the group consisting of B cell lymphoma, T cell lymphoma, leukemia, and multiple myeloma. Mixing and culturing the alginate solution and the cells;
Adding a collagen solution to the cultured mixture to prevent cell detachment;
Adding an agarose solution to the collagen-containing mixture, allowing the mixture to stand and gel, and culturing the cells three-dimensionally; And
And analyzing the growth, proliferation, death, differentiation and migration of the three-dimensional cultured cells. Mixing and culturing the alginate solution and the cells;
Adding a collagen solution to the cultured mixture to prevent cell detachment;
Adding an agarose solution to the collagen-containing mixture, allowing the mixture to stand and gel, and culturing the cells three-dimensionally; And
And analyzing the cell survival rate by adding an anticancer agent to the three-dimensional cultured cells.

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