Matrix for three-dimensional cell culture and application thereof

Abstract

The invention discloses application of a Dow Corning SYLGARD 184 organic silicon elastomer in preparation of a three-dimensional cell culture medium, and belongs to the technical field of biological materials. The matrix is formed by mixing an A-agent prepolymer and a B-agent curing agent of SYLGARD 184 according to the weight ratio of (8-12): 1 and then curing at the temperature of 35-39 DEG C. The method is characterized in that the substrate can support cell attachment without plasma treatment, and surface modification can be carried out through I-type collagen to enhance cell adhesion, so that a unique dual-mode cell microenvironment regulation and control system is formed. The matrix provided by the invention is simple to operate, low in cost and high in batch stability, effectively overcomes the defects of uncertain components, complex hydrogel synthesis treatment and the like of the existing animal-derived matrix, and can be widely applied to three-dimensional cell culture, disease model construction and drug screening platforms.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a matrix for three-dimensional cell culture and application thereof, and more particularly to the application of YLGARD 184 (hereinafter referred to as DC184) organic silicone elastomer material in the preparation of a three-dimensional cell culture matrix. BACKGROUND

[0002] Three-dimensional (3D) cell culture technology can significantly overcome the distortion of cell behavior, gene expression and drug response in traditional two-dimensional (2D) culture by simulating the in-vivo cell microenvironment, cell-cell interaction and biochemical signal gradient in a three-dimensional space, and has become a core platform in the fields of tissue engineering, disease modeling and drug screening. The realization of this technology highly depends on three-dimensional culture matrices that can simulate the in-vivo extracellular matrix (ECM). Currently, the three-dimensional culture matrices on the market are mainly divided into two categories: animal-derived matrices and synthetic polymer matrices.

[0003] 1. Prior art one: animal-derived matrix (represented by Matrigel)

[0004] Matrigel is a matrix extracted from the basement membrane of Engelbreth-Holm-Swarm (EHS) mouse sarcoma, rich in biological active ingredients such as laminin, collagen IV, and nidogen. It is widely used due to its good biocompatibility and ability to promote cell differentiation and self-organization.

[0005] Defects of the prior art: First, Matrigel has complex and unclear composition, with significant batch-to-batch differences, which seriously affects the repeatability and standardization of experiments. Second, as an animal-derived product, it has potential immunogenicity and pathogen contamination risks. Third, Matrigel is expensive, has strict storage conditions (below -20℃), and has a relatively short shelf life, resulting in high use costs. These defects limit its use in scenarios that require high consistency and large-scale application, such as high-throughput drug screening.

[0006] 2. Prior art two: synthetic polymer matrix (represented by polyethylene glycol (PEG) hydrogel)

[0007] PEG hydrogel is a class of high molecular weight materials synthesized by chemical synthesis, which has the advantages of clear composition, precise control of physical and chemical properties (such as hardness, porosity), and high batch stability.

[0008] Defects of the prior art: However, PEG hydrogel usually needs ultraviolet (UV) irradiation or additional chemical cross-linking agent to solidify into shape. UV irradiation can cause damage to cells, while the residue of chemical cross-linking agent can introduce cytotoxicity. In addition, PEG itself is anti-adhesion, and must be modified by complex chemical modification (such as connecting RGD peptide and other adhesion peptide segments) to make cells adhere, which is complicated and requires high technical requirements for experimenters, and also increases time and economic cost.

[0009] 3. Prior art three: physical aggregation method (such as hanging drop method)

[0010] The scaffold-free culture technology such as hanging drop method aggregates cells to form three-dimensional spheroids by physical means, which is relatively simple to operate.

[0011] Defects of the prior art: The size of the spheroids formed by this method is uneven, and lacks the mechanical and biochemical signal support provided by ECM, which cannot simulate the complex interaction between cells and matrix, and its application range is limited.

[0012] In summary, there is an urgent need in the art for a new three-dimensional cell culture matrix that can balance biocompatibility, clear composition, simple operation, controllable cost and functional adjustability. The ideal technical solution should avoid batch differences and risks of animal-derived matrix, while avoiding complex and potentially toxic solidification processes and surface modification steps required for synthetic polymer matrix. Specifically, the following technical problems need to be solved: how to provide a three-dimensional cell culture platform that does not require complex treatment (such as plasma treatment or UV curing) to achieve cell adhesion, can be easily modified to regulate cell behavior, and has good batch stability and low cost. SUMMARY

[0013] The present application aims to overcome the above-mentioned defects of the existing three-dimensional culture matrix, and provides a three-dimensional cell culture matrix which is simple to operate, low in cost, high in batch stability, and can form a dual-mode cell microenvironment regulation system, and its application. The matrix can support cell adhesion without plasma treatment, and can further regulate cell behavior through simple collagen modification.

[0014] The present application provides a three-dimensional cell culture matrix, which is composed of SYLGARD 184 silicone elastomer material, and the matrix can support cell adhesion and three-dimensional growth without plasma treatment.

[0015] Preferably, the SYLGARD 184 silicone elastomer material is mixed by A agent prepolymer and B agent curing agent at a weight ratio of (8-12): 1, and then cured at 35-39°C to form.

[0016] Preferably, the weight ratio of the A agent prepolymer and the B agent curing agent is 10:1.

[0017] Preferably, the surface of the matrix is modified with an extracellular matrix protein.

[0018] Preferably, the extracellular matrix protein is collagen type I.

[0019] The present application also provides a method for constructing a dual-mode cell microenvironment regulation system, comprising the following steps: using the SYLGARD 184 three-dimensional cell culture matrix as the first mode to support cells to form three-dimensional aggregates; and modifying the surface of the SYLGARD 184 three-dimensional cell culture matrix with collagen type I as the second mode to enhance cell adhesion.

[0020] The present application also provides the use of the three-dimensional cell culture matrix as described above in the preparation of a cell three-dimensional culture, a disease model construction or a drug screening platform.

[0021] Preferably, the cells are colorectal adenocarcinoma cells.

[0022] Preferably, the colorectal adenocarcinoma cells are HT29 cell lines or SW620 cell lines.

[0023] The SYLGARD 184 organic silicone elastomer material of the present application is formed by mixing the A agent prepolymer with the B agent curing agent and then curing. The present inventors have found through in-depth research and creative labor that although the recommended standard ratio is 10:1, the weight ratio of the A agent to the B agent in the range of 8:1 to 12:1 can all be successfully cross-linked and cured at a condition of 35℃ to 39℃ to form an elastic matrix suitable for three-dimensional cell culture. When the proportion of the A agent is increased (such as 12:1), the cross-linking density of the matrix after curing is relatively reduced, and the hardness is slightly decreased, which can simulate a softer physiological tissue microenvironment (such as brain tissue, adipose tissue); and when the proportion of the B agent is increased (such as 8:1), the cross-linking density is increased, and the hardness of the matrix is increased, which can simulate the tissue environment with higher hardness such as bone and cartilage. This hardness adjustability is one of the advantages of the present application, which enables the matrix to adapt to different cell culture scenarios with different mechanical properties. The temperature range of 35℃ to 39℃ covers the standard cell culture condition (37℃) and its slight fluctuations allowed, ensuring that the curing process matches the biocompatibility requirements of cell culture, and the curing rate is appropriate in this temperature range, which can form a matrix with uniform structure and no internal defects. Therefore, the skilled person in the art can select the weight ratio in the range of 8:1 to 12:1 according to the suitable mechanical microenvironment of the target cultured cell type, and perform curing under the conventional cell culture condition of 35℃ to 39℃, which can all achieve the purpose of the present application. Preferably, the weight ratio is 10:1, and the curing temperature is 37℃. Under this condition, the mechanical properties of the material (as described in the examples) are the most stable and balanced, which is suitable for most types of cell culture.

[0024] Compared with the prior art, the present application has the following advantages:

[0025] (1) DC184 can be self-crosslinked and cured at 37℃ without UV light or chemical initiator, avoiding the risk of cytotoxicity and simplifying the operation process.

[0026] (2) DC184 is a commercial product, which is significantly cheaper than Matrigel and other animal-derived matrices; its chemical composition is clear, and the ratio is fixed, ensuring high batch-to-batch stability.

[0027] (3) The unmodified DC184 surface supports cell aggregation to form three-dimensional spheroids; after simple modification with type I collagen, it changes to a mode that promotes cell adhesion, and one matrix can adapt to the culture needs of multiple cells.

[0028] (3) The matrix has good biocompatibility, high light transmittance (beneficial to microscopic observation), and adjustable mechanical properties, meeting the requirements of long-term three-dimensional cell culture. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0030] Figure 1 For the comparison chart of microscope photos of HT29 cells in Example 1 cultured on different substrates for 24 hours, 48 hours and 96 hours.

[0031] Figure 2 For the comparison chart of microscope photos of SW620 cells in Example 2 cultured on different substrates for 24 hours, 48 hours and 96 hours. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present application clearer, the following will combine the drawings in the present application to clearly and completely describe the technical solutions in the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0033] Experimental materials

[0034] To enable those skilled in the art to reproduce the present application, the materials used and their sources are listed in detail below. Equivalent products from other manufacturers can also be used, but their effects need to be verified through experiments.

[0035] SYLGARD 184 silicone elastomer kit was purchased from Dow Corning, including A agent (prepolymer, base) and B agent (curing agent), with a package specification of 100 g (A agent) + 10 g (B agent).

[0036] Type I rat tail collagen (Collagen I) was purchased from Corning®, product number 354236, specification 100 mg.

[0037] Phosphate buffered saline solution (PBS, 1x), pH 7.4, was purchased from Admas Company, instant powder, prepared as a 1 L solution according to the instructions.

[0038] Cell lines human colorectal adenocarcinoma cells HT29, SW620 were purchased from the American Type Culture Collection (ATCC).

[0039] Cell culture medium DMEM high-sugar medium, added with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-glutamine mixed solution (100X).

[0040] Experimental consumables 12-well cell culture plates, sterile syringes, centrifuge tubes, etc.

[0041] Example 1: Preparation of SYLGARD 184 (DC184) three-dimensional culture matrix

[0042] This example details the preparation process of the DC184 matrix.

[0043] Two sterile syringes were used to measure 10.0 grams of A agent and 1.0 gram of B agent of SYLGARD 184, respectively, and injected into a sterile centrifuge tube, ensuring a weight ratio of 10:1.

[0044] A sterile pipette tip or a small spatula was used to thoroughly mix the mixture for at least 3 minutes until it was evenly mixed and there were no obvious streaks. Then, the centrifuge tube was placed in a centrifuge and centrifuged at a speed of 1000 rpm for 1 minute to remove the air bubbles introduced during mixing, obtaining a clear and bubble-free prepolymer mixture.

[0045] The prepolymer mixture obtained in step 2 was taken up with a syringe and accurately added to each well of a 12-well cell culture plate at 1.3 milliliters. The culture plate was gently shaken to evenly cover the well bottom with the liquid.

[0046] Place the mixed mixture-coated plates into a cell culture incubator and let them cure for 48 hours at 37°C, 5% CO2, and saturated humidity. After the curing process is completed, the matrix forms a transparent, elastic solid film that tightly adheres to the bottom of the well plate. At this point, the matrix is ready for use in subsequent experiments or modifications.

[0047] This example further demonstrates the feasibility of forming a matrix from A agent and B agent at different ratios and temperatures, and illustrates the tunability of the mechanical properties. According to the weight ratios shown in the table below, use a sterile syringe to accurately measure the A agent and B agent of SYLGARD 184, and prepare a total of 5 groups of samples.

[0048]

[0049] After each group of mixed mixtures is thoroughly stirred and centrifuged to remove bubbles, they are injected into multiple 12-well plates. Place the well plates in incubators (or ovens with comparable temperature control accuracy) at 35°C, 36°C, 37°C, 38°C, and 39°C, respectively, and cure for 48 hours.

[0050] Result observation and evaluation: All 5 groups of ratios successfully cured into transparent, elastic solid films at 5 temperature points, indicating that the curing reaction can be effectively completed within the specified range of ratios and temperatures. The cured matrix is tested for hardness using a micro-press or similar device. It can be observed that as the proportion of B agent increases (from 12:1 to 8:1), the Young's modulus or Shore hardness of the matrix increases. The hardness of the 10:1 ratio is about the corresponding bone / cartilage level, while the 12:1 ratio is softer and the 8:1 ratio is harder. This proves that the mechanical properties of the matrix can be customized within a certain range by adjusting the ratio. Since SYLGARD 184 itself is biocompatible and the curing process is a physical crosslinking process without toxic byproducts, it can be reasonably expected that all matrices prepared within the above parameter range will have basic biocompatibility to support cell growth. The specific cell behavior regulation effect (such as spheroid formation or adherent growth) can be verified by cell experiments as described in Examples 3 and 4. This example demonstrates that the weight ratio of A agent to B agent is between 8:1 and 12:1, and the curing temperature is between 35°C and 39°C, and a structurally complete and mechanically adjustable SYLGARD 184 three-dimensional cell culture matrix can be successfully prepared. This provides direct support for the range described in the claims.

[0051] Example 2: Collagen Type I Modification of SYLGARD 184 Matrix

[0052] This example illustrates how to modify the surface of the DC184 matrix prepared in Example 1.

[0053] Under sterile conditions, the stock solution of type I rat tail collagen (concentration about 3-4 mg / mL) was diluted with pre-chilled PBS buffer to prepare a working solution of collagen at a concentration of 0.05 mg / mL. The whole operation was performed on ice to prevent premature gelation of the collagen.

[0054] To the solidified DC184 matrix in the 12-well plate in Example 1, 1 mL of the above collagen working solution was added to each well, ensuring that the liquid completely covered the surface of the matrix.

[0055] The culture plate was placed in a 37°C cell culture incubator and incubated for 30 minutes. During this period, the collagen molecules were immobilized on the surface of the DC184 matrix by physical adsorption.

[0056] After 30 minutes, the culture plate was removed from the incubator and the collagen working solution in the wells was carefully aspirated. Then, 1 mL of sterile PBS buffer was added to each well, gently shaken and aspirated, and this washing step was repeated twice to remove the collagen that was not firmly adsorbed.

[0057] The collagen-modified DC184 matrix after washing was ready for cell seeding. If not used immediately, a small amount of culture medium was added to prevent drying and stored at 4°C for a short period.

[0058] Example 3: Three-dimensional culture of HT29 cells on DC184 matrix

[0059] This example verifies the effect of the DC184 matrix and its collagen-modified form on the three-dimensional culture of HT29 cells.

[0060] The HT29 cells in the logarithmic growth phase were digested with conventional trypsin, resuspended in complete culture medium, and counted. The cell density was adjusted to prepare a single cell suspension.

[0061] The cell suspension was seeded into three different 12-well plates:

[0062] Control group: ordinary cell culture plate (TCP) without any treatment.

[0063] Experimental group B: plate with pure DC184 matrix prepared in Example 1.

[0064] Experimental group C: plate with collagen-modified DC184 matrix prepared in Example 2.

[0065] The number of cells seeded in each well was 150,000, and the final volume of culture medium in each well was 1 mL.

[0066] The culture plates inoculated with cells were placed in an incubator at 37°C, 5% CO2 for culture. Half of the old medium was replaced every 48 hours to maintain the nutrient supply. At 24 hours, 48 hours and 96 hours after inoculation, the growth morphology of cells in each well was observed and photographed using an inverted phase contrast microscope (corresponding to Figure 1 ).

[0067] The results show that in the control group (TCP), HT29 cells grow in a monolayer adherent manner with flat and spread morphology, showing typical two-dimensional culture characteristics. In the experimental group B (pure DC184), HT29 cells fail to adhere closely to the substrate surface, but instead aggregate with each other to form regular and compact three-dimensional spheroids. In the experimental group C (collagen-modified DC184), due to the enhanced adhesion of collagen, cells adhere to the substrate surface for growth, but the cell morphology is obviously rounded and three-dimensional, showing a three-dimensional morphology different from two-dimensional adhesion, but without forming discrete spheroids.

[0068] The results show that the pure DC184 substrate can effectively promote the formation of three-dimensional spheroids by HT29 cells; and after being modified by collagen, it supports the three-dimensional growth of cells in an adherent mode. This proves that the DC184 substrate provides a controllable dual-mode cell microenvironment system.

[0069] Example 4: Three-dimensional culture of SW620 cells on DC184 substrate

[0070] This example verifies the culture effect of DC184 substrate on another kind of colorectal cancer cell SW620, and the method is the same as that in Example 3. According to the steps in Example 3, 150,000 SW620 cells were inoculated in the control group, the pure DC184 group and the collagen-modified DC184 group. The observation results correspond to Figure 2 .

[0071] The results show that in the control group (TCP), SW620 cells grow in a monolayer adherent manner. In the experimental group B (pure DC184), SW620 cells first aggregate to form cell clusters, and then the cell clusters further expand and infiltrate to form a special three-dimensional structure with obvious circular holes. In the experimental group C (collagen-modified DC184), cells adhere to the substrate surface, and the morphology is more spread, and fail to form cell clusters and hole structures as shown in the experimental group B.

[0072] The results again confirm that the DC184 substrate supports the three-dimensional growth of SW620 cells, and the structure characteristics formed by it are different from those of HT29 cells, showing cell type specificity. Collagen modification also changes the growth behavior of cells. This shows that the substrate provided by the present application has good universality and controllability.

[0073] The above merely describes the preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application. Any person skilled in the art, without departing from the technical scheme of the present application, can make some changes or modifications to the above disclosed technical content to make equivalent embodiments with equivalent changes, but as long as it does not deviate from the technical scheme of the present application, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application are still within the scope of the technical scheme of the present application.

Claims

1. A three-dimensional cell culture substrate, characterized in that, The substrate is composed of SYLGARD 184 silicone elastomer material, which supports cell attachment and three-dimensional growth without plasma treatment.

2. The three-dimensional cell culture matrix according to claim 1, characterized in that, The SYLGARD 184 silicone elastomer material is formed by mixing A agent prepolymer and B agent curing agent at a weight ratio of (8-12):1, and curing at 35-39℃.

3. The three-dimensional cell culture matrix according to claim 2, characterized in that, The weight ratio of the A agent prepolymer and the B agent curing agent is 10:

1.

4. The three-dimensional cell culture matrix according to any one of claims 1 to 3, characterized in that, The surface of the substrate is modified by extracellular matrix protein.

5. The three-dimensional cell culture matrix according to claim 4, characterized in that, The extracellular matrix protein is collagen type I.

6. A method for constructing a dual-mode cell microenvironment regulation system, characterized in that, The method comprises the following steps: providing the SYLGARD 184 three-dimensional cell culture substrate as claimed in any one of claims 1 to 3 as a first mode to support cells to form three-dimensional aggregates; and modifying the surface of the SYLGARD 184 three-dimensional cell culture substrate with collagen type I as a second mode to enhance cell adhesion.

7. Use of the three-dimensional cell culture substrate as claimed in any one of claims 1 to 5 in the preparation of a three-dimensional cell culture, a disease model construction or a drug screening platform.

8. Use according to claim 7, characterized in that, The cells are colorectal adenocarcinoma cells.

9. Use according to claim 8, characterized in that, The colorectal adenocarcinoma cells are HT29 cell lines or SW620 cell lines.

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