Establishment method of high and low liver cancer matrix hardness experimental culture system

Abstract

The invention relates to the field of bioengineering, and discloses an establishment method of a high and low liver cancer matrix hardness experimental culture system. The method comprises the following steps: synthesis of RGD-SA, preparation of a CaCl2 solution, preparation of RGD-SA hydrogel, cell culture and recovery of RGD-SA hydrogel cells. According to the experimental culture system, on the basis that sodium alginate and calcium ions are subjected to chemical reaction to form calcium alginate, the hardness of three-dimensional hydrogel is adjusted by adjusting the concentration of the calcium ions, meanwhile, RGD is used for modifying SA so as to enhance and optimize the adhesion and hardness sensing ability of cells to the hydrogel, liver cancer cells are cultured in an RGD-SA hydrogel system, and the survival rate of the liver cancer cells is increased. In-vitro simulation of three-dimensional growth states of liver cancer cells in normal-hardness liver and high-hardness liver environments is well realized, and an ideal novel experimental research platform is provided for understanding liver cancer pathological characteristics and analyzing a hardness mechanical induction signal transmission process mechanism from the biomechanical perspective.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, more particularly, it relates to a method for establishing a high and low liver cancer matrix stiffness experimental culture system. BACKGROUND

[0002] Increased matrix stiffness is a significant biomechanical feature of most solid tumors, and the excessive deposition and cross-linking of matrix proteins are the main causes of increased ECM stiffness. Matrix stiffness mechanical signals can independently regulate tumor occurrence and progression. However, the in vitro simulation and related regulation of matrix stiffness are still in the initial stage. The culture vessels used for cell culture are mostly non-physiological hard materials such as polystyrene and glass, and the stiffness level of the culture substrate is much higher than the stiffness level of the tissue where the tumor is located. At the same time, the cells cultured in the culture vessels often exhibit abnormal behavior, including morphological flattening, abnormal polarization, changes in drug response, and loss of differentiation phenotype, which makes the cultured cells greatly different from the real pathological environment of the cells in vivo, especially the inability to simulate the real tissue stiffness level of the tumor tissue, making it difficult to use for stiffness mechanical signal regulation research. In the early stage, our research group formed a different stiffness substrate cell culture platform by changing the cross-linking degree of polyacrylamide gel, which better solved the problem of lack of experimental system for studying the mechanism of matrix stiffness regulation of liver cancer metastasis. Using different stiffness substrate cell culture platforms, we obtained a new mechanism that increased matrix stiffness promotes liver cancer cell migration and epithelial-mesenchymal transition, and found that glucose enrichment is a new feature of liver cancer lung premetastatic niche formation. However, the stiffness mechanical stimulation received by the cells cultured on the different stiffness substrate cell culture platform is mainly on the substrate, which belongs to two-dimensional cell culture, while the in vivo tumor cells are in an environment that not only receives signals in a single direction, but also receives stiffness mechanical signals in multiple dimensions. Therefore, further simulation of a three-dimensional matrix stiffness cell culture system can truly reflect the stiffness growth environment of tumor cells to solve the shortcomings and gaps of two-dimensional culture mode in reproducing the real environment of tumor cells.

[0003] Hydrogel is a polymer material with a three-dimensional network structure, which can be formed by chemical cross-linking, physical cross-linking and radiation cross-linking of polymers. Currently, there are many natural and synthetic polymers, such as chitosan, sodium alginate, collagen and polyvinyl alcohol. Among the current commercial three-dimensional culture hydrogels, collagen is the main component. In vitro collagen can provide good biocompatibility and cell adhesion for cells. However, the stiffness of the hydrogel formed by collagen is low, which often cannot accurately reflect and simulate the in vivo liver physiological and pathological stiffness environment.

[0004] Sodium alginate and calcium ion chelation to form hydrogel has good biocompatibility. The internal structure of sodium alginate hydrogel presents an eggshell-like structure, which does not change with the change of calcium ion concentration. The hardness of the hydrogel is linearly related to the concentration of calcium ions. By using this special cross-linking relationship, the hardness of the hydrogel can be independently adjusted without affecting other physical properties. And the hardness range of sodium alginate hydrogel is wide, which can be used to accurately simulate the physiological and pathological hardness conditions of liver in vivo.

[0005] In recent years, organoid culture plays an important role in tumor mechanism and drug research, and Matrigel is a widely used basic material for organoid culture. Due to the presence of various undefined components in Matrigel, it is difficult to study the influence of extracellular matrix hardness on organoid tumor characteristics by controlling its mechanical properties. At the same time, it is limited to improve the hardness level of Matrigel by supplementing collagen, and it cannot reach the tissue hardness of pathological liver. In the prior art, some researchers based on activated transglutaminase factor XIIIa (FXIIIa) enzyme cross-linking to generate inert polyethylene glycol (PEG) hydrogel to construct a hydrogel simulating the hardness of liver matrix. Although this hydrogel can show good biocompatibility and hardness sensitivity, the adjustable hardness range of this hydrogel is only 0.3kPa to 4kPa. As found in previous studies, the hardness of normal human liver is about 6kPa, and the hardness of cirrhotic liver is about 16kPa, which is far apart. Therefore, this hydrogel cannot ideally simulate the hardness environment of human liver tissue.

[0006] Therefore, there is an urgent need in the art for a high and low liver cancer matrix hardness experimental culture method that can more realistically simulate the hardness environment of human liver tissue. SUMMARY

[0007] The purpose of the application of the present application is to solve the problems existing in the prior art, such as the inability to accurately simulate the physiological and pathological hardness conditions of liver in vivo, and the limited adjustable hardness range.

[0008] In order to achieve the above-mentioned purpose of the application, the hydrogel based on sodium alginate has the advantages of wide adjustable matrix hardness range, linearly controllable hardness, convenient configuration, and recyclable cell clusters, and the like. The high and low liver cancer matrix hardness experimental culture system is successfully established, the hardness of the hydrogel can be independently adjusted, and at the same time, other physical properties such as biocompatibility and cell adhesion are not affected, so as to more accurately reflect the real growth environment of tumor cells in vivo, and to provide a more effective experimental platform for liver cancer mechanism research and drug screening.

[0009] Specifically, the present application provides the following technical solutions: In a first aspect, the present application provides a method for establishing a high and low liver cancer matrix hardness experimental culture system, comprising the following steps: Step 1, synthesis of RGD-SA Step 2, configuration of CaCl2 solution; Step 3, configuration of RGD-SA hydrogel and cell culture; Step 4, cell recovery of RGD-SA hydrogel.

[0010] Further, the step 1 comprises: Dissolve sodium alginate in 1M MES, then add NHS and EDC solution, continuously stir to activate the carboxyl group of sodium alginate; then add RGD freeze-dried powder, continuously stir overnight; then transfer the liquid into dialysis bag for dialysis, collect the solution in the dialysis bag after dialysis, filter the obtained liquid to remove bacteria, and freeze-drying preservation.

[0011] Further, in the step 1, the feeding ratio of sodium alginate, 1M MES, NHS, EDC and RGD is 1g: 100mL: 274mg: 484mg: 100mg.

[0012] Further, in the step 1, the dialysis process is as follows: first, cut the dialysis bag to an appropriate size, immerse the dialysis bag in a container containing an appropriate volume of dialysis bag treatment solution containing 2% NaHCO3 and 1 mM EDTA, and heat in a boiling water bath for 10 minutes; take out the dialysis bag and immerse it in ddH2O for 1-2 minutes, then immerse it in a container containing 1 mM EDTA solution, and heat in a boiling water bath for 10 minutes again; after cooling, wash with distilled water, then transfer the sodium alginate into a 3500 MW dialysis bag for dialysis for 3 days, and change ddH2O twice a day.

[0013] Further, the step 2 comprises: Dissolve CaCl2 powder in calcium ion-free DMEM, filter to remove bacteria, and store at 4°C.

[0014] Further, the step 3 comprises: Configure 4% RGD-SA with calcium ion-free DMEM, and resuspend the liver cancer cells with DMEM containing 80% FBS; add different volumes of CaCl2 solution according to the required hardness, and culture in a 37°C incubator; then aspirate the remaining liquid, and add DMEM medium containing 10% FBS for culture.

[0015] Further, in the step 3, the RGD-SA and the liver cancer cell suspension are mixed in a volume ratio of 4:1, so that the liver cancer cells are maintained at 1-4×10 5 / mL.

[0016] Further, the step 3, resuspension with 80% FBS DMEM refers to: trypsin digestion of cells, termination of digestion with 10% FBS DMEM, 1000 rpm centrifugation for 5 min, PBS washing of cells twice after removal of supernatant, 1000 rpm, 5 min; resuspension of cells with 80% FBS DMEM after removal of supernatant.

[0017] Further, the step 4 comprises: The RGD-SA hydrogel: EDTA = 1:5 ratio, 0.5M EDTA is added, 4°C shaking dissolution, 4°C, 3000 rpm centrifugation for 5 min, and the supernatant is discarded to obtain the cell precipitate.

[0018] In the second aspect, the application provides a high and low liver cancer matrix stiffness experimental culture system obtained by the establishment method.

[0019] Compared with the prior art, the application has the following beneficial effects: (1) The research on the regulation of the malignant characteristics of liver cancer cells based on the matrix stiffness of the different stiffness substrates can better solve the problem of the development of the stiffness regulation research, but due to the fact that the stiffness substrate cell culture platform can only simulate the single-dimensional stiffness environment of the growth of liver cancer cells in vitro, there is still a certain difference between the three-dimensional space stiffness environment of the liver cancer cells in the tumor tissue, and the three-dimensional stiffness environment of the liver cancer cells cannot be simulated. The establishment method of the liver cancer matrix stiffness three-dimensional culture experimental system can better simulate the pathological stiffness environment of the liver cancer tissue in vivo, reproduce the three-dimensional growth state of the liver cancer cells in the high and low matrix stiffness environment, and be used for exploring the influence and mechanism of the stiffness mechanical signal on the proliferation, invasion and drug resistance of the liver cancer cells in vitro.

[0020] (2) The novel liver cancer three-dimensional culture experimental system established by the application has the advantages of low cost, simple operation, stable experimental system and remarkable effect, and solves the technical problem that there is no ideal three-dimensional matrix stiffness experimental system for the stiffness mechanical research of liver cancer, and has good economic and application value. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the construction mode diagram of the high and low liver cancer matrix stiffness experimental culture system in the examples and the effect of different calcium ions on the morphology and stiffness of the hydrogel. Among them: (A), the flowchart of the construction of the liver cancer matrix stiffness experimental culture system; (B), the appearance of the hydrogel with different matrix stiffness; (C), the Young's modulus of the surface and the interior of the experimental system with different matrix stiffness detected by the indentation instrument.

[0022] Figure 2(A), SEM detection of surface pore size of high and low liver cancer matrix stiffness experimental system (hydrogel); (B), schematic diagram of hydrogel permeation experiment; (C), fluorescence intensity of lower chamber of high and low stiffness hydrogel at 0 min, 30 min, 60 min, 360 min, 720 min and 1440 min time points detected by permeation experiment.

[0023] Figure 3 (A), microscope shooting of shape and size of liver cancer cell clusters of SA, 3RGD-SA, 9RGD-SA modified high and low stiffness hydrogel at Day1, Day3, Day7, Day10, Day14; (B), schematic diagram of hydrogel permeation experiment; (C), fluorescence intensity of lower chamber of high and low stiffness hydrogel at 0 min, 30 min, 60 min, 360 min, 720 min and 1440 min time points detected by permeation experiment.

[0024] Figure 4 (A), qPCR detection of ICAM1 expression level of liver cancer cell clusters of SA, 3RGD-SA, 9RGD-SA modified high and low stiffness hydrogel; (B), qPCR detection of ITGB1 expression level of liver cancer cell clusters of SA, 3RGD-SA, 9RGD-SA modified high and low stiffness hydrogel; (C), qPCR detection of Paxillin expression level of liver cancer cell clusters of SA, 3RGD-SA, 9RGD-SA modified high and low stiffness hydrogel; (D), qPCR detection of Ki67 expression level of liver cancer cell clusters of SA, 3RGD-SA, 9RGD-SA modified high and low stiffness hydrogel; (E), qPCR detection of MMP2 expression level of liver cancer cell clusters of SA, 3RGD-SA, 9RGD-SA modified high and low stiffness hydrogel; (F), HE staining and immunofluorescence detection of YAP, Paxillin, ICAM1, KI67 expression level and localization of liver cancer cell clusters of SA, 3RGD-SA, 9RGD-SA modified high and low stiffness hydrogel. DETAILED DESCRIPTION

[0025] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be described clearly and completely below with reference to the drawings of the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the described embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without any inventive effort fall within the protection scope of the present application.

[0026] Example 1: Establishment of high and low liver cancer matrix stiffness experimental culture system The specific steps are as follows: Step 1, RGD-SA synthesis: dilute 1M MES (pH 6.5) to 0.1M MES (pH 6.5) with deionized water. Then take 1 g of sodium alginate and add it to 100 mL of the above prepared MES (0.1 M) and stir until fully dissolved. Then add 274 mg of NHS and 484 mg of EDC and continue stirring for 2 hours to activate the carboxyl group of sodium alginate. Then add 100 mg of RGD (3RGD: RGD; 9RGD: GGGGRGDSP) freeze-dried powder and continue stirring overnight to obtain an RGD-SA solution. Cut a 3500 MW dialysis bag of appropriate size (10-20 cm), immerse the dialysis bag in a container containing an appropriate volume of dialysis bag treatment solution (2% NaHCO3, 1 mM EDTA), and heat in a boiling water bath for 10 minutes. Take out the dialysis bag and immerse it in ddH2O for 1-2 minutes, then immerse it in a container containing 1 mM EDTA (pH 8.0) solution, and heat in a boiling water bath for 10 minutes again. Then transfer the RGD-SA into the 3500 MW dialysis bag and dialyze for 3 days, changing the ddH2O twice a day. Collect the solution in the dialysis bag, filter the obtained liquid to remove bacteria, and then freeze-dry using a freeze dryer.

[0027] Step 2, 1M CaCl2 solution preparation: weigh 4.4394 g of CaCl2 powder and dissolve it in 40 mL of calcium-free DMEM, and filter to remove bacteria.

[0028] Step 3, RGD-SA hydrogel preparation and cell culture process: prepare 4% RGD-SA using calcium-free DMEM medium and shake overnight at 37°C. Digest the cells using trypsin and terminate the digestion using 10% FBS DMEM, centrifuge at 1000 rpm for 5 min, wash the cells twice with PBS at 1000 rpm for 5 min, and resuspend the cells in 80% FBS DMEM after removing the supernatant. Mix the RGD-SA and cell suspension at a ratio of 4:1 (volume ratio). The number of cells is maintained at 1-4 x 10 5Mix the solution gently at a concentration of 1 mL, avoiding the formation of air bubbles. Then add the mixture to the well plate and add calcium ion solutions of different concentrations (20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, all of which were obtained by diluting with 1 M CaCl2 solution). Incubate at 37°C for 1.5 h. Figure 1 A is a schematic diagram of the culture system for liver cancer matrix stiffness experiment. Figure 1 B represents the appearance of hydrogels with different matrix hardness. To test the hardness, the obtained hydrogels were tested using an indenter to examine the surface and internal hardness. Figure 1 C represents the surface and internal Young's modulus of different matrix stiffness experimental systems. Based on physiological and pathological liver stiffness, 25 mM calcium ion concentration sodium alginate hydrogel simulates normal liver stiffness, and 45 mM calcium ion concentration sodium alginate hydrogel simulates high liver stiffness. Residual liquid was aspirated, and DMEM medium (containing 10% FBS) was added. The next day, DMEM medium (containing 10% FBS) was replenished as needed. After 48 hours, the medium was replaced with 50-80% DMEM.

[0029] Step 4, RGD-SA hydrogel cell recovery: Remove the hydrogel and place it in a sterile centrifuge tube. Wash twice with PBS, 5 min each time. Then add 0.5 M EDTA at a hydrogel:EDTA ratio of 1:5 and shake at 4°C for 20 min. Centrifuge at 4°C, 3000 rpm for 5 min, discard the supernatant to obtain the cell pellet.

[0030] Example 2: Surface pore size and permeability of high and low liver cancer matrix stiffness experimental systems Scanning electron microscopy was used to examine the surface pore structure of high and low hepatocellular carcinoma matrix stiffness experimental systems (hydrogels) to clarify the relationship between surface pore size and permeability of hydrogels with different stiffness. The results showed that both high and low stiffness hydrogels formed an eggshell-like structure, and there was no significant difference in pore size between hydrogels of different stiffness. Figure 2 A. The permeability of the high and low hepatocellular carcinoma matrix stiffness experimental systems (hydrogels) was analyzed using a dextran permeation experiment, as shown in Figure 2B. The results showed no significant difference in permeability between the high and low hepatocellular carcinoma matrix stiffness experimental systems (hydrogels). Figure 2 C. The above results indicate that the high and low hepatocellular carcinoma matrix stiffness experimental systems (hydrogels) have uniform structure and permeability, and can be used to investigate the influence of matrix stiffness on the malignant biological behavior of hepatocellular carcinoma cells.

[0031] Example 3: Experimental systems for high and low hepatocellular carcinoma matrix stiffness (hydrogels) to investigate the effects of different modification methods and different stiffness on cell cluster proliferation and survival rate. After preparing a novel experimental system (hydrogel) for high and low hepatocellular carcinoma matrix stiffness, cells were cultured, and cell images were taken under a microscope on days 1, 3, 5, 7, 10, and 14 after cell culture. Figure 3 A), at 14 days, cell clusters were stained using a live / dead cell staining kit, and high-content imaging and 3D reconstruction were performed. Figure 3 B, Figure 3 (C) indicates that different modification methods and different hardness have no significant effect on cell cluster activity.

[0032] Example 4: Analysis of the tissue formation tendency and malignant characteristics of liver cancer cell clusters cultured using high and low liver cancer matrix stiffness experimental systems (hydrogels) After preparing experimental systems (hydrogels) with different modified high and low hepatocellular carcinoma matrix stiffness, cells were cultured for 14 days, and cell clusters were collected from the hydrogels. The expression of ICAM1, ITGB1, PXN, Ki-67, and MMP2 in the cell clusters cultured with different modifications was detected by qPCR. The results showed that 9RGD-modified SA promoted cell cluster adhesion and proliferation more effectively than 3RGD-modified SA and unmodified SA, exhibiting a more pronounced tendency towards tissue formation. Simultaneously, the high-sceleonocellular carcinoma matrix stiffness group of hydrogel-cultured hepatocellular carcinoma cell clusters also showed significantly increased malignant characteristics such as adhesion, proliferation, and metastasis. Figure 4 A-4E). Immunofluorescence was used to detect the expression of ICAM1, PXN, Ki-67, and YAP in cell clusters cultured with different modifications. The results showed that hepatocyte clusters formed within the 9RGD-SA high-stiffness hepatocellular carcinoma matrix hydrogel better promoted cell-cell and cell-hydrogel adhesion, exhibited a more significant tissue formation trend, and promoted cell proliferation, invasion, and metastasis. It could also effectively sense and transmit stiffness mechanical signals, simulating hepatocellular carcinoma tissues with different stiffness backgrounds, and was used to analyze and explore the influence of hepatocellular carcinoma matrix stiffness on the malignant biological behavior of hepatocellular carcinoma cells. Figure 4 F).

[0033] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for establishing a high and low liver cancer matrix stiffness experimental culture system, characterized in that, The method comprises the following steps: Step 1, synthesis of RGD-SA Step 2, preparation of CaCl2 solution; Step 3, preparation of RGD-SA hydrogel and cell culture; Step 4, recovery of cells in RGD-SA hydrogel.

2. The establishment method according to claim 1, characterized by, The step 1 comprises: Dissolve sodium alginate in 1M MES, then add NHS and EDC solution, continuously stir to activate the carboxyl group of sodium alginate; then add RGD freeze-dried powder, continuously stir overnight; then transfer the liquid into a dialysis bag for dialysis, collect the solution in the dialysis bag after dialysis, filter the obtained liquid to remove bacteria, and freeze-dry for storage.

3. The establishment method according to claim 2, characterized in that, In the step 1, the feeding ratio of sodium alginate, 1M MES, NHS, EDC and RGD is 1g:100mL:274mg:484mg:100mg.

4. The establishment method according to claim 2, characterized in that, In the step 1, the dialysis process is as follows: first, cut the dialysis bag to an appropriate size, immerse the dialysis bag in a container containing an appropriate volume of dialysis bag treatment solution containing 2% NaHCO3 and 1mM EDTA, and heat in a boiling water bath for 10 minutes; take out the dialysis bag and immerse it in ddH2O for 1-2 minutes, then immerse it in a container containing 1mM EDTA solution, and heat in a boiling water bath for 10 minutes again; after cooling, wash with distilled water until the sodium alginate is transferred into a 3500 MW dialysis bag for dialysis for 3 days, and change ddH2O twice a day.

5. The method of establishing of claim 1, wherein, The step 2 comprises: Dissolve CaCl2 powder in calcium ion-free DMEM, filter to remove bacteria, and store at 4°C.

6. The establishment method of claim 1, wherein, The step 3 comprises: Prepare 4% RGD-SA with calcium ion-free DMEM, and resuspend liver cancer cells with DMEM containing 80% FBS; add different volumes of CaCl2 solution according to the required hardness, and culture in a 37°C incubator; then aspirate the remaining liquid, and add DMEM medium containing 10% FBS for culture.

7. The establishment method according to claim 6, characterized by, In step 3, the RGD-SA is mixed with the liver cancer cell suspension at a volume ratio of 4:1, so that the liver cancer cells are maintained at a concentration of 1-4 x 10 5 / mL.

8. The establishment method according to claim 6, characterized by, In the step 3, the step of resuspending with DMEM containing 80% FBS refers to: digesting the cells with trypsin, stopping the digestion with 10% FBS DMEM, centrifuging at 1000 rpm for 5 min, washing the cells twice with PBS at 1000 rpm for 5 min, and resuspending the cells with 80% FBS DMEM after removing the supernatant.

9. The establishment method of claim 1, wherein, The step 4 comprises: Add 0.5M EDTA to RGD-SA hydrogel:EDTA=1:5, dissolve at 4°C on a shaker, centrifuge at 4°C and 3000 rpm for 5 min, and discard the supernatant to obtain cell pellets.

10. A high or low liver cancer matrix hardness experimental culture system obtained by the method of any one of claims 1-9.

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