Efficient transient transfection method for human primary cartilage cells based on electroporation

Abstract

The invention discloses an efficient transient transfection method for human primary chondrocytes based on electroporation, and belongs to the field of cell transfection, and the method comprises the following specific steps: resuspending the human primary chondrocytes in an electrotransfection mixed solution, sucking a resuspension solution with a set volume, carrying out primary electroporation under the conditions that the pulse intensity is 550V and the pulse duration is 20ms, and carrying out secondary electroporation to obtain the human primary chondrocytes. Then, transferring the electroporated cells into a preheated culture medium for culturing, so that efficient instantaneous transfection can be realized; as a rapid, efficient, stable and convenient primary cell transient transfection method, the problems of low cell transfection efficiency, unknown electroporation parameters, low cell survival rate and the like caused by a cartilage extracellular matrix barrier effect in the prior art are solved, and the most basic conditions are provided for carrying out functional experiments in primary cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell transfection, and in particular to a high-efficiency transient transfection method for human primary chondrocytes based on electroporation. BACKGROUND

[0002] Transfection is an important technology for introducing exogenous genes into cells, which enables eukaryotic cells to actively or passively receive DNA, RNA and other nucleic acid molecules under certain conditions, so that they can be expressed in cells, thereby realizing the regulation and research of cell function. With the continuous development of life science, transfection has become one of the routine tools for studying gene function, disease mechanism and assisting gene therapy, and also provides a basis for cell therapy, tissue engineering and biopharmaceuticals, and promotes the development of cell engineering field. The continuous innovation and optimization of transfection technology also provide more possibilities for research in the field of life science. Current transfection methods can be divided into stable transfection and transient transfection. Stable transfection can integrate exogenous genes into the cell genome, and after drug screening, a long-term stable modified cell line can be obtained, which is usually achieved by viral infection. However, this method is complex to operate and has a long experimental period. If you want to quickly detect the expression effect of exogenous genes in a short period of time, transient transfection is the best choice. Transient transfection can efficiently express recombinant DNA in a short period of time, which is usually achieved by chemical transfection methods such as high-molecular polymer transfection, liposome transfection or physical transfection methods such as electroporation transfection, etc.

[0003] Primary cells are cells directly isolated from living tissues or organs for culture. Compared with cell lines, primary cells have morphological structure and physiological function closer to the original tissue in vivo, and are more ideal biological models. However, the transfection efficiency of primary cells is generally low, which is related to their physiological state, cell membrane characteristics and culture conditions, etc. In addition, cell type also greatly affects the transfection efficiency. Especially for primary chondrocytes, human chondrocytes will secrete matrix macromolecules during monolayer culture, resulting in accumulation of a large amount of extracellular matrix in the culture medium, so it is difficult to transfect them efficiently. For primary chondrocytes, slow virus delivery method is currently used at home and abroad, but this method has high experimental cost and long cycle, usually taking 1-2 weeks, which cannot realize short-term and rapid expression of genes. Therefore, it is necessary to develop a simple and easy high-efficiency transient transfection method according to the physiological characteristics of primary chondrocytes. SUMMARY

[0004] In the prior art, there are problems of low cell transfection efficiency, unknown electroporation parameters and low cell survival rate caused by the extracellular matrix barrier effect of chondrocytes. Therefore, in order to solve the above problems, the present application provides a high-efficiency transient transfection method for human primary chondrocytes based on electroporation.

[0005] In order to achieve the above object, the present application provides the following technical scheme: a high-efficiency transient transfection method of human primary chondrocytes based on electroporation, and the specific steps are as follows: The human primary chondrocytes are resuspended in an electroporation mixed solution, and a set volume of the resuspension is sucked to perform electroporation to realize cell electroporation. The cells after electroporation are transferred into a preheated culture medium to realize high-efficiency transient transfection.

[0006] Further, the human primary chondrocytes are primary chondrocytes separated from the cartilage tissue of a patient with osteoarthritis.

[0007] Further, the configuration ratio of the electroporation mixed solution is electroporation buffer Part A: Part B: plasmid = 5:5:1, wherein the plasmid is a pCMV-copGFP plasmid, and the pCMV-copGFP plasmid is extracted by using an endotoxin-free plasmid small extraction kit.

[0008] Further, before the human primary chondrocytes are resuspended in the electroporation mixed solution, the following treatment is performed: When the cell density of the human primary chondrocytes in the culture dish reaches 90%, trypsin digestion is added, and after the cell morphology is round, complete culture medium is added to stop digestion, and after centrifugation to remove the supernatant, the cells are gently resuspended in serum-free DMEM / F12 culture medium, centrifuged, and the primary chondrocyte precipitate is obtained.

[0009] Further, 1×10 5 The human primary chondrocytes are resuspended in the electroporation mixed solution, a set volume of the resuspension is sucked into an electroporation tube, and the Celetrix electroporation instrument is used for electroporation.

[0010] Further, when the Celetrix electroporation instrument is used for electroporation, the electroporation is performed once, the pulse intensity is 480 V-550 V, and the pulse time is 20 ms-30 ms.

[0011] Further, after the electroporation tube body is moistened, a set volume of the resuspension is added from the bottom of the electroporation tube.

[0012] Further, after the resuspension is added to the electroporation tube, there is no air bubble in the electroporation tube, and the cells are uniformly distributed.

[0013] Further, the preheated culture medium is specifically an anti-free DMEM / F12 culture medium containing 10% fetal bovine serum, and the preheating temperature is 37℃.

[0014] Further, the cells after electroporation are transferred into the preheated culture medium to culture for 48 h.

[0015] Compared with the prior art, the present application has at least the following beneficial effects: The application provides a high-efficiency transient transfection method of human primary chondrocytes based on electroporation, which overcomes the extracellular matrix barrier effect of chondrocytes by using the electroporation method, directly penetrates the extracellular matrix and cell membrane of chondrocytes by electric shock, realizes gene introduction, avoids the transfection difficulty problem caused by the extracellular matrix barrier in the traditional transfection technology, and significantly improves the transfection efficiency of primary chondrocytes. Further, the application optimizes the proportion of the electroporation mixed solution and the electric shock parameters, ensures the effectiveness and stability of the electroporation, reduces the damage of electric shock to cells, reduces cell apoptosis, improves the survival rate of cells, and ensures that the cells after transfection have good survival ability. Further, the application uses the pCMV-copGFP plasmid extracted by the endotoxin-free plasmid small extraction kit, avoids the immune response caused by the traditional virus vector or chemical reagent, and improves the safety and reliability of transfection. Further, the application optimizes the electroporation steps, ensures the health and activity of primary chondrocytes at different stages, and provides the best basic conditions for subsequent functional experiments. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The transfection effect comparison of different chemical reagent transfection methods (high molecular polymer transfection method PEI MAX, HighGene plus and liposome transfection reagent Lipo8000 TM , TransIntro® PL) in human primary chondrocytes is presented, and the microscope magnification in the picture is 10 (eyepiece) x 4 (objective).

[0017] Figure 2 The transfection of human primary chondrocytes in the electroporation experiment is presented, and the microscope magnification in the picture is 10 (eyepiece) x 4 (objective).

[0018] Figure 3 The influence of different pulse times on the survival rate of primary chondrocytes in the electroporation experiment is presented, and the microscope magnification in the picture is 10 (eyepiece) x 4 (objective).

[0019] Figure 4 The influence of different pulse intensities on the transfection efficiency of primary chondrocytes in the electroporation experiment is presented, and the microscope magnification in the picture is 10 (eyepiece) x 4 (objective).

[0020] Figure 5 The flow chart of transfecting human primary chondrocytes by different methods is provided. DETAILED DESCRIPTION

[0021] For more clearly, intuitively present the technical solutions in the application and its key steps, the following content will combine the drawings, the specific embodiments of the application are described in detail. Obviously, the described embodiments are only a part of the embodiments of the application rather than all, based on the embodiments in the application, all other embodiments obtained by those skilled in the art after simple condition replacement or deduction, belong to the protection scope of the application.

[0022] As Figure 5 shown, the application provides a high-efficiency transient transfection method of human primary chondrocytes based on electroporation by comparing with other transfection methods and optimizing the experimental parameters of electroporation, including the following steps: Step one: pCMV-copGFP plasmid extraction Step two: isolation and culture of human primary chondrocytes Step three: transfection of human primary chondrocytes using different methods 1. Chemical transfection: using high molecular polymer transfection reagent and liposome transfection reagent, transfecting human primary chondrocytes according to the proportion suggested in the product instruction.

[0023] 2. Electroporation: using Celetrix electroporator to transfect human primary chondrocytes.

[0024] After 48 hours of transfection, the expression level of green fluorescent protein is observed under fluorescence microscope, so as to characterize the transfection efficiency of different methods.

[0025] Step four: optimizing the experimental parameters of primary chondrocyte electroporation to improve the transient transfection efficiency.

[0026] The product information of reagents, consumables and instruments involved in the embodiments is shown in Table 1.

[0027] Table 1 Reagents, consumables and instruments

[0028] The detailed implementation steps of the application are as follows: Step one: pCMV-copGFP plasmid extraction The pCMV-copGFP plasmid is introduced into the DH5a competent cells by heat shock transformation, and after adding the LB liquid medium without antibiotics, it is recovered in the bacterial incubator at 37℃, 200 rpm for one hour, inoculated into the LB liquid medium containing Kana resistance, and cultured overnight at 37℃, 200 rpm, and the pCMV-copGFP plasmid is extracted using the endotoxin-free plasmid small extraction kit.

[0029] Step two: isolation and culture of human primary chondrocytes Human cartilage tissue was obtained from osteoarthritis patients undergoing total knee replacement with the approval of the ethics committee. The cartilage tissue was cut into pieces using surgical scissors and washed with resistant PBS, then the supernatant was discarded, 0.25% trypsin working solution was added and digested for half an hour at 37°C in a 180 rpm shaker, resistant PBS was added for blowing and sucking, and the cartilage pieces were transferred to 0.2% collagenase II working solution and digested for 14 to 16 hours at 37°C in a 150 rpm shaker. The digestion was terminated by adding fetal bovine serum, and the cells were filtered through a 70 μm cell filter, and the filtrate was centrifuged at 300 x g for 5 minutes. The cell pellet was resuspended in DMEM / F12 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and placed in a cell culture incubator. The medium was changed once every 3-5 days, and the cells were subcultured when the cell density reached more than 90%.

[0030] Step three: Transfection of human primary chondrocytes using different methods 1. Chemical transfection: Two different high-molecular polymer transfection reagents and liposome transfection reagents were tested respectively.

[0031] 1) Cell plating: Human primary chondrocytes were cultured in a 100 mm cell culture dish. When the cell density reached 80%-90%, the cells were trypsinized and centrifuged at 1000 rpm for 5 minutes to collect the cells. The cells were inoculated into a 12-well plate at a ratio of 1:1, and the 12-well plate was shaken to distribute the cells evenly. The plate was placed in a cell culture incubator overnight.

[0032] 2) Preparation of transfection mixture: The transfection mixture was prepared according to the ratio of 1 μg plasmid per well, plasmid: transfection reagent = 1:2 and 1:4. Specifically, for 12-well plate cells, the transfection mixture was prepared according to 1 μg pCMV-copGFP plasmid + 100 μL Opti-MEM + 2 / 4 μL transfection reagent per well. The mixture was gently blown and mixed and incubated at room temperature for 15 minutes. The specific steps are as follows: Prepare 8 1.5 mL centrifuge tubes, add 100 μL Opti-MEM and 1 μg plasmid to each tube, mix well, then add 2 / 4 μL high-molecular polymer transfection reagent PEI MAX (Yesen), HighGene plus Transfection reagent (ABclone) and liposome transfection reagent Lipo8000 TM (Biyun Tian), TransIntro PL (full type gold). After gentle blowing and mixing, incubate at room temperature for 15 minutes.

[0033] 3) Replace the 12-well plate cells with 10% fetal bovine serum-free DMEM / F12 medium, and inoculate the incubated transfection mixture into each well of the cells, and place them in a cell culture incubator. After 6 hours, replace the complete medium containing double antibodies.

[0034] 4) After 48 hours of culture, observe the expression of green fluorescent protein under a fluorescence microscope. According to the contrast between bright field and dark field, reflect the transfection efficiency under different transfection reagents.

[0035] The experimental results are shown in Figure 1 As shown in the figure, in human primary chondrocytes, the expression level of green fluorescent protein corresponding to the high molecular polymer transfection method is slightly higher than that of the liposome transfection method, but overall, the transfection efficiency of the two methods is poor, that is, the transfection reagent fails to efficiently carry the plasmid through the extracellular matrix of chondrocytes, and the liposome transfection method shows significant cytotoxicity, with a large number of cell deaths, which indicates that the chemical reagent transfection method is not suitable for this cell.

[0036] 2. Electroporation transfection: use Celetrix electroporator to transfect human primary chondrocytes.

[0037] When performing electroporation transfection test, the cell concentration is preferably 5×10 6 cells / mL, and the configuration ratio of the electroporation mixture is electroporation buffer Part A:Part B:plasmid=5:5:1 (v:v:m). Specifically, 20 μL of the electroporation mixture corresponds to 1×10 5 cells of primary chondrocytes, and the electroporation mixture is 10 μL of electroporation buffer Part A + 10 μL of Part B + 2 μg of pCMV-copGFP plasmid, which is gently blown and mixed. The electroporation buffer Part A, Part B and pCMV-copGFP plasmid are mixed to prepare the electroporation mixture. After washing and centrifugation of the trypsin-digested primary chondrocytes, the electroporation mixture is used for resuspension, and then transferred into a 20 μL electroporation tube and immediately placed into a Celetrix electroporator (CTX-1500A). After setting the pulse intensity and pulse time, electroporation is performed. Then the cells are transferred to a preheated cell culture medium for culture for 48 hours. The specific steps are as follows: 1) Culture human primary chondrocytes in a 100 mm cell culture dish. When the cell density reaches 90%, electroporation transfection can be performed.

[0038] 2) Prepare an anti-10% fetal bovine serum-containing DMEM / F12 culture medium in advance and preheat it to 37°C in a cell culture incubator.

[0039] 3) According to the 20 μL electroporation tube specification, take 10 μL of electroporation buffer Part A and Part B respectively, and add 2 μg of pCMV-copGFP plasmid, and gently blow and mix to obtain the electroporation mixture. Avoid generating bubbles during the process.

[0040] 4) Add 1 mL trypsin to 100 mm cell culture dish, let it stand for 3-5 minutes at 37°C, and then observe the cell morphology under a microscope. When the cells are round, add 1-2 mL of complete medium and blow and suck several times, then centrifuge at 1000 rpm for 5 minutes. Use a pipette to completely aspirate the supernatant, collect about 1 x 10 5 primary chondrocyte pellet, and avoid affecting the effect of electroporation.

[0041] 5) When performing electroporation transfection experiments, gently resuspend the primary chondrocyte cells after centrifugation twice with the electroporation mixture. Use the electroporation gun head to aspirate 20 μL of liquid, insert the electroporation gun head into the 20 μL electroporation tube body and rotate the electroporation tube to wet the tube body. Then insert the gun head into the tube bottom and slowly add the liquid. Throughout the process, do not move the position of the pipette. Stop after the pipette is set to the first gear and remove the gun head. The whole process should be as fast as possible to avoid bubbles and cell sedimentation in the electroporation tube during liquid transfer, which will affect the electroporation effect.

[0042] 6) Turn on the Celetrix electroporator in advance and set the pulse intensity to 500 V and the pulse time to 20 ms. After the electroporation tube is ready, quickly place it in the electroporator and perform a single electroporation. Transfer the electroporated cells to the preheated medium in 2) and shake evenly before placing in the incubator for culture.

[0043] 7) After 48 hours of transfection, observe the green fluorescent protein expression level under a fluorescence microscope. According to the bright-dark field contrast, reflect the transfection efficiency of electroporation.

[0044] The experimental results are shown in Figure 2 . As shown in the figure, when the pulse intensity is 500 V and the pulse time is 20 ms, the electroporation transfection method shows high transfection efficiency in human primary chondrocytes, indicating that electroporation can effectively penetrate the extracellular matrix barrier, and the cell viability after transfection is high and the state is good, making it an ideal method for primary cell transfection.

[0045] Step four: optimization of electroporation experiment parameters This part is the optimization of electroporation experiment parameters of human primary chondrocytes. The detailed experimental operation is shown in step three 2. Electroporation.

[0046] 1. Test the cell viability of human primary chondrocytes after electroporation when the pulse intensity is 480 V and the pulse time is 20 ms, 25 ms and 30 ms, respectively.

[0047] The experimental results are shown in Figure 3From the microscope, it can be clearly observed that as the pulse time is prolonged from 20 ms to 25 ms, 30 ms, the number of adherent cells gradually decreases, and the pulse time of 20 ms ensures the survival rate of most cells, and therefore the pulse time of 20 ms is selected for subsequent experiments.

[0048] 2. Test the transfection efficiency of human primary cells after electroporation when the pulse time is 20 ms and the pulse intensity is 480 V, 520 V and 550 V, respectively.

[0049] The experimental results are shown in Figure 4 As shown in the figure, as the pulse intensity increases, the expression level of green fluorescent protein in human primary chondrocytes also increases, and the cell density remains basically unchanged, indicating that the cell is not sensitive to the pulse voltage, and the current can penetrate the extracellular matrix and cell membrane barrier in the range of 480 V to 550 V, realizing effective delivery of the plasmid.

[0050] In summary, we obtain an efficient and reliable electroporation transfection method and parameter conditions suitable for human primary chondrocytes, 1x10 5 primary chondrocytes are added to a 20 muL electric shock tube, the pulse intensity of Celetrix electric transfection instrument is set to 550 V, the pulse time is 20 ms, and the electric shock is performed once, so that the best transient transfection effect can be obtained.

[0051] The primary chondrocytes targeted by the present application secrete a large amount of extracellular matrix (such as type II collagen, proteoglycan, etc.), which may hinder the contact of the chemical transfection reagent with the cell membrane, resulting in low transfection efficiency. The electroporation transfection utilizes the current to break through the cell membrane to promote the entry of DNA molecules into the cell, and generally exhibits fewer restriction conditions and higher transfection efficiency. However, there is no research showing that electroporation can overcome the barrier effect of the extracellular matrix to achieve high-efficiency transfection. In addition, the experimental parameters of electroporation are also important parameters for determining the transfection effect. Too high pulse intensity and too long pulse time will cause great damage to the cells, resulting in a large number of cell deaths, and vice versa. The extracellular matrix and cell membrane of the chondrocytes cannot be penetrated to realize effective delivery of DNA. In summary, the present application compares the chemical transfection methods such as high-molecular polymer transfection, liposome transfection and electroporation transfection, and proves that the chemical transfection method cannot successfully pass through the extracellular matrix barrier to complete the transfection of chondrocytes, while the electroporation method is simple and easy to operate. Through multiple adjustments of the experimental parameters, the plasmid can be efficiently delivered to the target cells by penetrating the extracellular matrix, and the transfection toxicity is low and the cell survival rate is high, which provides the most basic conditions for the smooth progress of the primary chondrocyte function experiment, and also provides a feasible idea for the transfection of cells with extracellular matrix barrier effect.

[0052] The application provides a high-efficiency transient transfection method of human primary chondrocytes based on electroporation, the electroporation method can penetrate the extracellular matrix of the chondrocytes, and excellent transfection effect is shown; meanwhile, the best electroporation experiment parameters for the primary chondrocytes are given, and the most basic conditions for various functional experiments in the primary chondrocytes are provided. The application provides a high-efficiency, simple and high-cell-viability transfection method by optimizing the electroporation experiment steps and experiment parameters, and overcomes the technical difficulty that the existence of the extracellular matrix of the chondrocytes leads to low cell transfection efficiency.

Claims

1. A method for efficient transient transfection of human primary chondrocytes based on electroporation, characterized in that, The specific steps are as follows: The human primary chondrocytes are resuspended in the electrotransformation mixture, and a set volume of the resuspension is taken for electroshock to realize cell electroporation; The cells after electroporation are transferred into the preheated culture medium for culture to realize high-efficiency transient transfection.

2. The method of claim 1, wherein the method is a high efficiency transient transfection of human primary chondrocytes based on electroporation. The human primary chondrocytes are primary chondrocytes isolated from the cartilage tissue of a patient with osteoarthritis.

3. The method of claim 1, wherein the method is a high efficiency transient transfection of human primary chondrocytes based on electroporation. The configuration ratio of the electrotransformation mixture is electrotransformation buffer Part A: Part B: plasmid = 5:5:1, wherein, The plasmid is a pCMV-copGFP plasmid, and the pCMV-copGFP plasmid is extracted by using an endotoxin-free plasmid small extraction kit.

4. The method of claim 1, wherein the method is a high efficiency transient transfection of human primary chondrocytes based on electroporation. Before the human primary chondrocytes are resuspended in the electrotransformation mixture, the following treatment is performed: When the cell density of the human primary chondrocytes in the culture dish reaches 90%, trypsin digestion is added, and after the cell morphology becomes round, complete culture medium is added to stop the digestion, and after centrifugation to remove the supernatant, the cells are gently resuspended in serum-free DMEM / F12 culture medium, centrifuged, and the primary chondrocyte precipitate is obtained.

5. The method of claim 1, wherein the method is a high efficiency transient transfection of human primary chondrocytes based on electroporation. 1 x 10 5 The primary chondrocytes were resuspended in the electrotransformation mix and a set volume of the resuspension was pipetted into the cuvette and electroporated using the Celetrix electroporator.

6. The method of claim 5, wherein the method is a high efficiency transient transfection of human primary chondrocytes based on electroporation. When the Celetrix electrotransformation instrument is used for electroshock, the shock is once, the pulse intensity is 480 V-550 V, and the pulse time is 20 ms-30 ms.

7. The method of claim 5, wherein the method is a high efficiency transient transfection of human primary chondrocytes based on electroporation. After the shock tube body is moistened, a set volume of resuspension is added from the bottom of the shock tube.

8. The method of claim 5, wherein the method is a high efficiency transient transfection of human primary chondrocytes based on electroporation. After the resuspension is added to the shock tube, no air bubbles are required in the shock tube, and the cells are uniformly distributed.

9. The method of claim 1, wherein the method is a high efficiency transient transfection of human primary chondrocytes based on electroporation. The preheated culture medium is specifically an anti-10% fetal bovine serum-containing DMEM / F12 culture medium, and the preheating temperature is 37℃.

10. The method of claim 1, wherein the method is a high efficiency transient transfection of human primary chondrocytes based on electroporation. The cells after electroporation are transferred into the preheated culture medium for culture for 48 h.

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