Pelletizing agent, application thereof and method for constructing three-dimensional cell model
By using sphere-forming agents of matrix gel, methyl cellulose and collagen in three-dimensional cell culture, combined with the hanging drop method, the problem of cells not easily aggregation and low spherical rate is solved, and a three-dimensional cell model with uniform size and dense size is achieved. Suitable for research on various cell types and drug development.
Patent Information
- Application Number
- CN202411346863.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2024-12-24
AI Technical Summary
In the existing three-dimensional cell culture technology, cells are not easy to aggregate, have low spherical yields, inconsistent sizes of cell spherical cells, and require consumables such as well plates to affect the experimental efficiency and controllability of the results.
Using a ball-forming agent containing matrix gel, methyl cellulose and collagen, combined with the hanging drop method, cells are cultured in conventional laboratory culture dishes, adjust the concentration and proportion of the ball-forming agent, promote cell aggregation and ball-forming, and achieve uniform size. , dense and high spherical rate three-dimensional cell model.
It has achieved a high sphere-forming rate of cell balls (98%-100%), uniform size, dense structure, and high cell viability. It is suitable for a variety of cell types and is suitable for in vitro solid tumor construction and drug development.
Smart Images
Figure CN119177209A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of cell culture, in particular to a spheroidizing agent and its application and a method for constructing a three-dimensional cell model. Background Art
[0002] Three-dimensional cell culture refers to the technology of growing cells in artificially created three-dimensional spatial structures, which enables cells to migrate and grow in three-dimensional spatial structures. The three-dimensional culture method better simulates the natural microenvironment of cell survival in organisms, allowing cells to present more realistic biochemical and physiological responses under controllable culture conditions.
[0003] The hanging drop method is a common 3D cell culture method, which uses the characteristics of cells self-aggregating into spheroids to construct a three-dimensional cell culture model. Usually, the hanging droplets are placed in a special plate with an open bottomless well, and the three-dimensional cell spheroids cultured in the hanging drop plate for a few days can simulate the microenvironment of tumor cells, in which the oxygen, nutrients required by the cells and the metabolic waste produced can enter or be sent out in a gradient, forming a physiological state similar to that in the body. However, due to the differences in the growth states of different cells, some cells are not easy to aggregate or agglomerate; or even if they are spheroidized, the cell spheroids are of different sizes, poor consistency, and a low proportion of spheroids, and are relatively loose. Therefore, the present invention intends to develop a spheroidizing agent, combined with the hanging drop method, without the use of consumables such as well plates, using conventional laboratory culture dishes, a three-dimensional cell model with uniform size, good consistency, dense structure, high spheroidization rate, controllable model size, stable model and good cell survival rate can be prepared. Summary of the invention
[0004] The purpose of the present invention is to provide a spheroidizing agent and its application and a method for constructing a three-dimensional cell model to solve the problems existing in the above-mentioned prior art. The present invention provides a spheroidizing agent, which, combined with the hanging drop method, can culture three-dimensional cell spheres using conventional laboratory culture dishes without the aid of consumables such as well plates, and has a high spheroidization rate; at the same time, the spheroidizing agent solves the problems of difficult cell aggregation, difficult agglomeration, and uneven size of cell spheres.
[0005] To achieve the above object, the present invention provides the following solutions:
[0006] The invention provides a spheroidizing agent, which comprises Matrigel, methylcellulose and collagen.
[0007] Preferably, the volume-to-mass ratio of matrix gel, methylcellulose and collagen in the spheronizing agent is 0.01-2 mL: 24 g: 0.5-5 mg.
[0008] Preferably, the volume mass ratio of matrix gel, methyl cellulose and collagen in the spheronizing agent is 1 mL: 24 g: 1.2 mg.
[0009] The present invention provides the use of the spheroidizing agent in constructing a three-dimensional cell model.
[0010] The present invention provides a method for constructing a three-dimensional cell model, comprising the following steps:
[0011] The cell suspension is mixed with the above-mentioned spheroidizing agent to obtain a mixed solution; the volume percentage of matrix gel in the mixed solution is 0.01-2%, the concentration of methylcellulose is 0.24 g / mL, and the concentration of collagen is 5-50 μg / mL;
[0012] The mixed solution is dropped onto a culture plate and cultured to obtain a three-dimensional cell model.
[0013] Preferably, the culture condition parameters are: culturing in an incubator at 37° C. and 5% CO 2 for 24-72 hours; and semi-quantitative fluid replacement every 24 hours.
[0014] Preferably, the concentration of cells in the cell suspension is 0.3×10 6 / mL-1×10 6 Pieces / mL.
[0015] Further preferably, the cells include Hacat cells, Raw264.7 cells, HFF-1 cells and MCF-10A cells.
[0016] The present invention provides a three-dimensional cell model constructed by using the above construction method.
[0017] The present invention provides the use of the above three-dimensional cell model in one or more of the following (1)-(6):
[0018] (1) Application in drug development;
[0019] (2) Application in drug screening;
[0020] (3) Application in drug testing;
[0021] (4) Application in drug testing;
[0022] (5) Application in constructing pharmacological and pathological tumor models;
[0023] (6) Application in simulating in vitro solid tumor models.
[0024] The present invention provides the use of the spheroidizing agent in improving the spheroidization rate of three-dimensional cells.
[0025] The present invention discloses the following technical effects:
[0026] The invention provides a spheroidizing agent. When constructing a three-dimensional cell model, by adding Matrigel, collagen and methylcellulose at specific concentrations, cells are more easily aggregated, the spheroidizing rate is high and can reach 98%-100%, and the cell spheres are uniform in size; at the same time, the spheroidizing agent is suitable for a variety of cells.
[0027] The present invention also provides a method for constructing a three-dimensional cell model, which combines the spheroidizing agent provided by the present invention with the hanging drop method to construct a three-dimensional cell sphere; the cell sphere has uniform size, good consistency, dense structure, high spheroidization rate, controllable model size, stable model and good cell viability, and can be applied to in vitro solid tumor construction, drug development, screening and detection and other research; at the same time, by adjusting the cell amount, three-dimensional cell models of different sizes can be constructed in vitro, and the method is simple to operate. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0029] Figure 1 The effect of different addition amounts of substances in the spheroidizing agent on cell spheroidization; among them, ad is the spheroidizing effect of spheroidizing agent 1 to spheroidizing agent 4, and the scale bars in ad are all 432.9 μm;
[0030] Figure 2 The three-dimensional cell spheres of different sizes were constructed; ac was the cell concentration of 3×10 5 / mL, 5×10 5 / mL and 7×10 5 The three-dimensional cell model was constructed with a cell suspension of 100 cells / mL; the scale bars in ac are 432.9 μm;
[0031] Figure 3 The three-dimensional spheres of different types of cells were constructed; a is MCF-10A cells; b is RAW264.7 cells; c is human keratinocytes; d is human fibroblasts; the scale bars in ad are 432.9 μm;
[0032] Figure 4 Figure 5 is the effect diagram of MCF-10A cell spheroidization. Figures a and b are 4 randomly selected MCF-10A cell spheroidization effects. The scale bars in figures a and b are 432.9 μm. DETAILED DESCRIPTION
[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.
[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0038] Example 1 Two-dimensional culture of cells
[0039] Human keratinocytes Hacat and mouse macrophages Raw264.7 were cultured in 10% DMEM complete medium in a 37°C incubator with 5% CO2. When the cell density reached 80%, they were digested and passaged using trypsin.
[0040] Human foreskin fibroblasts HFF-1 were cultured in 15% DMEM complete medium in a 37°C incubator with 5% CO2. When the cell density reached 80%, trypsin was used for digestion and passage.
[0041] Normal human mammary epithelial cells MCF-10A were cultured in DMEM / F12 (1:1) medium + insulin (10 μg / mL) + epidermal growth factor (20 ng / mL) + 100 ng / mL cholera virus + 0.5 μg / mL hydrocortisone + 5% horse serum medium in a 37°C incubator with 5% CO2. When the cell density reached 80%, trypsin was used for digestion and passage.
[0042] Example 2 Study on the effect of different amounts of substances added in the spheroidizing agent on cell spheroidization
[0043] The conditions for adding different substances to the spheroidizing agent were explored, and the cell amount was 5×10 5 A MCF-10A cell suspension of 10 μg / mL was mixed with a spheroidizing agent (to promote cell aggregation) to obtain a mixed solution, and then the mixed solution was dropped on a culture plate in an appropriate volume (10 μL) using the hanging drop method. The culture plate was turned over and placed on a water chamber (a 6 mm culture dish containing PBS) for culture. The culture condition parameters were semi-quantitative liquid replacement every 24 h, and 3D culture was obtained by culturing at 37°C for 48 h.
[0044] Among them, the spheroidizing agents are spheroidizing agents 1 to spheroidizing agents 4; spheroidizing agent 1 is matrigel, and the volume percentage of matrigel in the mixed solution (the mixed solution obtained by mixing MCF-10A cell suspension with the spheroidizing agent) is 2%; spheroidizing agent 2 is matrigel and collagen, the volume percentage of matrigel in the mixed solution is 2%, and the collagen is 45 μg / mL; spheroidizing agent 3 is matrigel, methyl cellulose and collagen, the volume percentage of matrigel in the mixed solution is 2%, the methyl cellulose is 0.24 g / mL, and the collagen is 12 μg / mL; spheroidizing agent 4 is matrigel, methyl cellulose and collagen, the volume percentage of matrigel in the mixed solution is 1%, the methyl cellulose is 0.24 g / mL, and the collagen is 12 μg / mL.
[0045] The effects of different amounts of substances added to the spheroidizing agent on cell spheroidization were then investigated. Figure 1 As shown, the spheroidizing effects of different spheroidizing agents are different, spheroidizing agent 3 and spheroidizing agent 4 have better effects, but spheroidizing agent 4 has the best effect. Therefore, spheroidizing agent 4 was selected for subsequent experiments.
[0046] Example 3 Study on the effect of different proportions of spheroidizing agents on cell spheroidization
[0047] The ratio of each component in the spheroidizing agent 4 was optimized. We selected MCF-10A cells and conducted three experiments in each group. Each time, 5×10 5Different proportions of spheroidizing agents were added to 1 mL of cell suspension with a concentration of 1 cell / mL, and cell spheroids (10 μL / spheroid) were prepared by the hanging drop method, and the cell spheroidization rate was statistically analyzed. The specific setting system is shown in Table 1.
[0048] Table 1 Statistical results of cell sphere formation rate
[0049]
[0050]
[0051] The results showed that the spheroidization rate was the highest when the volume percentage of matrigel in the mixed solution was 1%, methylcellulose was 0.24 g / mL, and collagen was 12 μg / mL.
[0052] Example 3 Construction of three-dimensional cell spheres of different sizes
[0053] Prepare cells at a concentration of 3 × 10 5 / mL, 5×10 5 / mL and 7×10 5 A MCF-10A cell suspension of 100 μg / mL was added with a spheroidizing agent 4 to obtain a mixed solution, in which the volume percentage of matrigel was 1%, methylcellulose was 0.24 g / mL, and collagen was 12 μg / mL. The mixed solution was then dropped onto a culture plate in an appropriate volume (10 μL) using the hanging drop method. The culture plate was flipped over and placed on a water chamber (6 mm culture dish containing PBS) for culture. The culture condition parameters were semi-quantitative liquid replacement every 24 hours, culture at 37°C for 48 hours, and three-dimensional cell spheres of different sizes were prepared. The results are shown in Figure 2. Figure 2 As shown, the three-dimensional cell sphere structures of different cell numbers are regular and the sphere-forming effect is good.
[0054] Example 4 Construction of three-dimensional spheres of different cell types
[0055] Human keratinocytes Hacat, mouse macrophages Raw264.7, human foreskin fibroblasts HFF-1, and human normal mammary epithelial cells MCF-10A were used to construct three-dimensional cell models of different cells according to the method of step Example 3, wherein the cell concentration in the cell suspension was 5×10 5 The results are as follows Figure 3 As shown, the three-dimensional cell spheres constructed by using the sphere-forming agent for different types of cells have regular structures and good sphere-forming effects.
[0056] Example 5 Statistics of cell spheroidization rate and spheroid size
[0057] MCF-10A cells were used to calculate the cell sphere formation rate and sphere size. Three experiments were performed, with 5×10 51 mL of cell suspension containing 10 μg / mL cells was added to the spheroidizing agent 4 to obtain a mixed solution, in which the volume percentage of matrigel was 1%, methylcellulose was 0.24 g / mL, and collagen was 12 μg / mL. 96 (the first time in Table 2), 98 (the second time in Table 2), and 101 droplets (the third time in Table 2) of 10 μL were prepared respectively, and then the mixed solution was dropped on the culture plate in a suitable size using the hanging drop method, and the spheroidization rate and size of the three-dimensional cell sphere were counted. The results are shown in Tables 2 and Figure 4 As shown, the cell sphere formation rate was above 98%, and the cell sphere size was in the range of 360 μm-400 μm.
[0058] Table 2 Statistical results of cell sphering rate
[0059] deal with Number of cell spheres Balling rate first 96 98.9％ Second time 98 100％ The third time 101 98％
[0060] In summary, the present invention uses a specific spheroidizing agent and a hanging drop method to construct a three-dimensional cell culture model, which solves the problems of difficult cell aggregation, difficult agglomeration, and uneven cell spheroid size; and the constructed three-dimensional cell spheroid model has uniform size, good consistency, dense structure, high spheroidization rate, controllable model size, stable model, and good cell viability. Therefore, the three-dimensional cell model constructed using the spheroidizing agent can be applied to in vitro solid tumor construction, drug development, screening, and detection research.
[0061] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A spheronizing agent, characterized in that: The spheronizing agent includes matrigel, methylcellulose and collagen.
2. The spheronizing agent according to claim 1, characterized in that The volume mass ratio of matrix gel, methyl cellulose and collagen in the spheroidizing agent is 0.01-2 mL: 24 g: 0.5-5 mg.
3. The spheronizing agent according to claim 1 or 2, characterized in that: The volume mass ratio of matrix gel, methyl cellulose and collagen in the spheronizing agent is 1 mL: 24 g: 1.2 mg.
4. Use of the spheronizing agent according to any one of claims 1 to 3 in constructing a three-dimensional cell model.
5. A method for constructing a three-dimensional cell model, characterized in that: The following steps are involved: Mixing the cell suspension with the spheroidizing agent according to any one of claims 1 to 3 to obtain a mixed solution; the volume percentage of matrix gel in the mixed solution is 0.01-2%, the concentration of methylcellulose is 0.24 g / mL, and the concentration of collagen is 5-50 μg / mL; The mixed solution is dropped onto a culture plate and cultured to obtain a three-dimensional cell model.
6. The construction method according to claim 5, characterized in that: The culture condition parameters are: culture in an incubator at 37° C. and 5% CO 2 for 24-72 hours; and semi-quantitative fluid replacement every 24 hours.
7. The construction method according to claim 5, characterized in that: The cell concentration in the cell suspension is 0.3×10 6 / mL-1×10 6 Pieces / mL.
8. A three-dimensional cell model constructed using the construction method described in any one of claims 5 to 7.
9. Use of the three-dimensional cell model according to claim 8 in one or more of the following (1)-(6): (1) Application in drug development; (2) Application in drug screening; (3) Application in drug testing; (4) Application in drug testing; (5) Application in constructing pharmacological and pathological tumor models; (6) Application in simulating in vitro solid tumor models.
10. Use of the spheroidizing agent according to any one of claims 1 to 3 in improving the spheroidization rate of three-dimensional cells.