Method for transfecting Chinese hamster ovary cells through electroporation

Abstract

The invention discloses a method for transfecting Chinese hamster ovary cells through electroporation, which comprises the following steps: unfreezing CHO cells, and transferring the cells into a CD303 culture medium for suspension growth; the method comprises the following steps: adding a culture medium into a cell culture bottle, taking cells, putting the cells into a centrifugal tube, centrifuging, discarding a supernatant, adding a PBS (Phosphate Buffer Solution), centrifuging, discarding the supernatant, adding the PBS in each reaction, and uniformly blowing and beating to form a cell suspension; adding plasmids into a centrifugal tube, adding a PBS (Phosphate Buffer Solution), uniformly mixing, and adding the plasmids into the cell suspension; sucking a mixed solution of the cells and plasmids, introducing recombinant plasmids into Chinese hamster ovary (CHO) cells by utilizing an electroporation transfection method, and carrying out an electroporation transfection test; cell counting and expression quantity detection are carried out after cell culture. According to the invention, plasmids of a target sequence are introduced into Chinese hamster ovary (CHO) cells by adopting an electroporation transfection method, the transfection efficiency is improved by optimizing cell transfection conditions, the recovery time of cell strains after electroporation is reduced, and the expression quantity and survival rate of the CHO cells after electroporation transfection are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a method for electrotransfecting Chinese hamster ovary cells. Background Art

[0002] Therapeutic antibodies are usually produced by highly expressing stable (Chinese hamster ovary cells CHO) cell lines. Currently, CHO cells have become a commercial production host due to their ease of use, established regulatory record, and safety. The glutamine synthetase (GS) gene expression system (GS-CHO expression system) is adopted. High-yield mammalian cell lines rely on efficient transfection and the production of stable cell populations. Increasing the expression level of transfected genes in Chinese hamster ovary CHO cells can reduce the culture volume during the production process, which is of great significance for biological research. Therefore, a method for electrotransfecting Chinese hamster ovary cells is designed to improve the transfection efficiency, aiming to reduce the recovery time after electropermeabilization of cell lines and increase the protein expression level.

[0003] It should be noted that the above introduction of the technical background is only for the convenience of clearly and completely explaining the technical solution of the present invention and facilitating the understanding of those skilled in the art. It cannot be considered that the above technical solutions are well-known to those skilled in the art just because these solutions are described in the background art part of the present invention. Summary of the Invention

[0004] To overcome the deficiencies in the above-mentioned prior art, the purpose of the present invention is to provide a method for electrotransfecting Chinese hamster ovary cells.

[0005] To achieve the above purpose and other related purposes, the technical solution provided by the present invention is: a method for electrotransfecting Chinese hamster ovary cells, comprising the following steps: Step 1: Preparation of cells: Thaw CHO cells, transfer the cells to CD303 medium containing L-Glutamine for suspension growth, and passage the cells for three generations. Step 2: Treatment of cells: Add fresh complete medium for cell passage and preheat it in a cell culture flask. Take CHO cells with a cell number of 0.5 - 2×10 7 and put them into a centrifuge tube. After centrifugation, discard the supernatant, add PBS solvent, after centrifugation, discard the supernatant, add PBS solvent to each reaction, and blow and beat evenly to form a cell suspension; for example, the cell number can be: 0.5×10 7 、1×10 7 、1.5×10 7 or 2×10 7 .

[0006] Step 3: Add 25 μg to 60 μg of plasmid into a centrifuge tube, gently mix after adding PBS solvent, and then add the plasmid into the cell suspension; aspirate the cell and plasmid mixture, and use the electroporation transfection method to introduce the recombinant plasmid into Chinese hamster ovary (CHO) cells, and optimize the amount of transfected cells; perform electroporation transfection tests on 0.5E7 to 2E7 cells; perform electroporation transfection tests on 25 μg to 60 μg of plasmid; for example, the plasmid amount can be: 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, or 60 μg.

[0007] Step 4: Respectively culture the samples after electroporation, and perform cell counting and expression level detection after culture.

[0008] Further, in Step 1, when preparing the cells, the CHO cells need to be placed in a 37 °C water bath for 2 to 3 minutes to completely dissolve the cells. For example, the cell water bath time can be 2 minutes, 2.5 minutes, or 3 minutes.

[0009] Further, in Step 1, add 4 mM L-Glutamine to the CD303 medium, and provide a 5% CO2 concentration and a suspension growth condition at 37 °C.

[0010] Further, in Step 2, the cell culture flask uses a T-25 cell culture flask and add 7 mL of fresh complete medium for cell passage into it. The complete medium contains 4 mM Glutamine, and the preheating condition is 37 °C.

[0011] Further, in Step 2, the rotation speed of the centrifugation treatment is 1000 rpm, and the time is 5 minutes; the addition amount of PBS solvent is 0.3 mL to 0.5 mL, and the cell suspension is placed at room temperature for less than or equal to 30 minutes. The cell suspension is placed at room temperature for no more than 30 minutes, which can avoid reducing cell activity and transfection efficiency. For example, the addition amount of PBS solvent is 0.3 mL, 0.4 mL, or 0.5 mL.

[0012] Further, in Step 3, the conditions of the electroporation are: pulse voltage 240 to 330 V, pulse time 20 to 30 ms, and the number of pulses is not less than once. For example, the pulse voltage is: 240 V, 270 V, 300 V, 330 V; the pulse time is: 20 ms, 25 ms, or 30 ms.

[0013] Further, the pulse voltage is 270 V, and the pulse time is 25 ms.

[0014] Further, in step three, the addition amount of the PBS solvent is 0.4 mL to 0.6 mL. For example, the addition amount of the PBS solvent is 0.4 mL, 0.5 mL or 0.6 mL.

[0015] Further, in step four, the cell culture time is not less than 24 h.

[0016] Further, the plasmid amount is 60 μg and the cell volume is 1.5E7.

[0017] Due to the application of the above technical solution, the beneficial effects of the present invention compared with the prior art are as follows: 1. The present invention adopts the Gene Pulser Xcell™ electroporation system. The Gene Pulser Xcell™ electroporation system is the latest generation of electroporation transfection system at present. Through the action of a high-intensity electric field, the permeability of the cell membrane is instantaneously increased, so as to absorb exogenous molecules in the surrounding medium. This electroporation technology is a valuable and effective alternative method compared with other physical and chemical transformation methods.

[0018] 2. The present invention applies the Gene Pulser Xcell™ electroporation system to introduce the target protein into Chinese hamster ovary CHO cells. On this basis, an innovative design is carried out on the electroporation conditions, which are the factors that have the greatest influence on the transfection efficiency of Chinese hamster ovary CHO cells by electroporation. The cell survival rate and expression level far exceeding those of physical and chemical transformation methods can be achieved, and the cell transfection efficiency is greatly improved.

[0019] 3. The electroporation transfection method for Chinese hamster ovary CHO cells of the present invention has no vector capacity limitation, solves the problem of the small capacity of the virus vector system, and can efficiently introduce exogenous genes within 20 kb into Chinese hamster ovary VHO cells. Research shows that genes longer than 20 kb can also be introduced by electroporation, but the transfection efficiency will fluctuate.

[0020] 4. The present invention directly transports genes into cells, avoiding risks such as adverse immune reactions, toxic effects and genetic defects caused by viral vector integration generated by viral vector systems and chemical transfection methods. It has high safety, simple operation and is easy to repeat; 5. The present invention uses electroporation transfection technology to transfer exogenous genes into human stem cells. The time required for effective transfection of cells is short, the expression time of exogenous genes is relatively fast, and high-efficiency expression of the target gene can be observed 24 hours after transfection, while it takes at least 72 hours to observe the expression of the target gene using the virus vector system. This greatly shortens the culture time after transfection and also improves the transfection efficiency. Description of the Drawings

[0021] Figure 1Detection graph of expression level after 24 hours in Example 1 of the present invention; Figure 2 Detection graph of cell density after 24 hours in Example 1 of the present invention; Figure 3 Detection graph of cell viability after 24 hours in Example 1 of the present invention; Figure 4 Detection graph of expression level after 24 hours in Example 2 of the present invention; Figure 5 Detection graph of cell density after 24 hours in Example 2 of the present invention; Figure 6 Detection graph of cell viability after 24 hours in Example 2 of the present invention; Figure 7 Detection graph of expression level after 24 hours in Example 3 of the present invention; Figure 8 Detection graph of cell density after 24 hours in Example 3 of the present invention; Figure 9 Detection graph of cell viability after 24 hours in Example 3 of the present invention; Figure 10 Detection graph of expression level after 24 hours in Example 4 of the present invention; Figure 11 Detection graph of cell density after 24 hours in Example 4 of the present invention; Figure 12 Detection graph of cell viability after 24 hours in Example 4 of the present invention; Detailed implementation manners To make the above objects, features and advantages of the present invention more apparent and understandable, the following provides a detailed description of the specific implementation manners of the present invention. Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0022] Unless otherwise indicated in the operating examples or otherwise stated, all numbers used in the specification and claims to represent amounts of ingredients, physical and chemical properties, etc. are understood to be adjusted by the term "about" in all cases. Therefore, unless otherwise stated, the numerical parameters listed in the above specification and appended claims are approximate values, and those skilled in the art can appropriately change these approximate values in seeking to obtain the desired characteristics using the teachings disclosed herein. The use of numerical ranges expressed by endpoints includes all numbers within that range and any range within that range. For example, 1 to 5 includes 1, 1.1, 1.3, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.

[0023] The following elaborates on the preferred embodiments of the present invention in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making the scope of protection of the present invention more clearly defined.

[0024] In the present invention, "L-Glutamine" refers to L-glutamine; "CD303 medium" refers to a specific medium used for culturing cells expressing CD303 (also known as BDCA-2); "PBS solvent" refers to phosphate-buffered saline solution, which may precipitate during storage, so it needs to be resuspended (i.e., the precipitated substances are redispersed in the liquid) during the experiments of the present invention; "CHO cells" are Chinese hamster ovary cells.

[0025] The plasmid in the present invention uses a GFP plasmid. The plasmid with a GFP tag can emit green fluorescence under ultraviolet light irradiation and is used for labeling and detection in biological research.

[0026] Under certain conditions, reducing the time constant can perform local and spatially precise electroporation on the target cells it captures and transports, minimizing the electrolysis of all cells in the chamber due to long-term exposure to the electric field, improving the cell viability after electroporation and thus enhancing the cell transfection efficiency. Under certain conditions, increasing the concentration of the transfected plasmid helps to improve the cell transfection efficiency.

[0027] When transfecting the target sequence into Chinese hamster ovary CHO cells by electroporation, the sample volume in the electroporation cuvette is inversely proportional to the sample resistance. Reducing the sample volume will increase the sample conductivity. By reducing the sample volume to change the sample resistance, the reversible electroporation yield is increased, and the effect of reducing the time constant is achieved. Compared with standard electroporation, because only a small part of the electrode plates play a role, the influence on the cell membrane in contact with the electrode plates is reduced.

[0028] The specific implementation process of the method for transfecting Chinese hamster ovary cells by electroporation: Preparation of cells: Thaw a vial of CHO cells from the liquid nitrogen tank, place it in a 37 °C water bath for 2.5 minutes. After the cells are completely dissolved, transfer the cells to InnoMedia CHO CD303 medium containing 4 mM L-Glutamine and grow in suspension under the conditions of 5% CO2 concentration and 37 °C. The cells are passaged every 2 - 3 days. After three passages, cell transfection operations are carried out.

[0029] Treatment of cells: On the day of cell transfection, add 7 mL of fresh complete medium for cell passage (containing 4 mM Glutamine) to the T-25 cell culture flask before electroporation and preheat it in a 37 °C cell incubator.

[0030] The number of cells to be transfected is 0.5 - 2×10 7 [Calculation method: The number of cells = density × volume. Specifically, the density of the cells to be transfected is measured as C1 (cells / mL), and V1 mL [V1 = (6.0 - 10.0×106) / C1] of CHO cells is aspirated into a 50 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, and the supernatant is discarded; Add 0.5 mL of PBS to resuspend the cell pellet, centrifuge at 1000 rpm for 5 min, and discard the supernatant; Add 0.3 mL of PBS to each reaction to resuspend the cell pellet, pipette evenly and then count the cells. The cell suspension should not be placed at room temperature for more than 30 minutes, otherwise it will lead to a decrease in cell viability and transfection efficiency.

[0031] Electroporation transfection operation: Add 25 μg of plasmid to a 1.5 mL centrifuge tube, add 0.5 mL of PBS, mix gently, then add the plasmid to the cell suspension. Aspirate the cell and plasmid mixture (make sure there are no air bubbles in the pipette tip) and add it to a 4 mm electroporation cuvette. Set the pulse voltage, pulse width, and number of pulses on the electroporator; then vertically insert the electroporation cuvette with the sample into the electroporator; select the electroporation program and press the Start key on the touch screen; record the actual electroporation parameter values displayed on the electronic display screen, transfer the pulsed sample to a culture flask containing pre-warmed medium that has been prepared, and place the culture plate in the incubator for culture; Among them, the method of introducing plasmids of different proteins into Chinese hamster ovary CHO cells: 1. Use the Gene Pulser Xcell™ electroporation system and introduce the recombinant plasmid into Chinese hamster ovary CHO cells by electroporation transfection; the amount of transfected cells was optimized, and 0.5E7 - 2E7 cells were tested for electroporation transfection. The conditions for electroporation were a pulse voltage of 240 - 330 V, a pulse duration of 20 - 30 ms, and at least one pulse; 2. Use the Gene Pulser Xcell™ electroporation system and introduce the recombinant plasmid into Chinese hamster ovary CHO cells by electroporation transfection; the amount of transfected plasmid was optimized, and 25 μg - 60 μg of plasmid was tested for electroporation transfection. The conditions for electroporation were a pulse voltage of 240 - 330 V, a pulse duration of 20 - 30 ms, and at least one pulse; 3. Culture the samples after electroporation respectively, and perform cell counting and expression level detection after culture.

[0032] 24 h after electroporation transfection: After culturing for 24 h, the cells were collected immediately for the detection of cell viability and expression level.

[0033] The transfection efficiency directly affects the protein expression level after the introduction of foreign genes. Establishing the optimal conditions for electroporation transfection to improve the transfection efficiency is crucial for enhancing the protein expression level in Chinese hamster ovary (CHO) cells. According to the existing technology, the factors known to affect electroporation transfection include: cell-related factors such as cell passage number, cell physiological state, cell density, etc.; plasmid concentration, purity, and transfection amount; electroporation parameters, namely pulse voltage, pulse duration, and pulse number, etc.

[0034] Example 1: Based on the above "specific implementation process", "cell number: 1.5×10 7 , plasmid amount: 40 μg, pulse voltage: 70 V, pulse times: 20 ms, 25 ms, 30 ms" were selected as the experimental conditions. After transfection with the expression plasmid containing the target gene, the transfection efficiency and cell viability after 24 h were detected. The expression level detection chart, cell density detection chart, and cell viability detection chart after 24 h of electroporation transfection are shown in Appendix Figure 1 Appendix Figure 2 Appendix Figure 3 respectively, and the relationship table of experimental conditions and "cell density after 24 h, cell viability after 24 h, expression level after 24 h" is as follows: Table 1

[0035] It can be seen from the data in Table 1 that under the same conditions, the longer the pulse time, the higher the protein expression level and the higher the electroporation efficiency. Considering the cell viability and expression level comprehensively, it is considered that the cell viability and expression level in the second group are both within a relatively appropriate range.

[0036] Example 2: Based on the above "specific implementation process", "cell numbers: 1×10 7 , 1.5×10 7 , 2×10 7 , plasmid amount: 40 μg, pulse voltage: 240 V, pulse time: 25 ms" were selected as the experimental conditions. After transfection with the expression plasmid containing the target gene, the transfection efficiency and cell viability after 24 h were detected. The expression level detection chart, cell density detection chart, and cell viability detection chart after 24 h of electroporation transfection are shown in Appendix Figure 4 Appendix Figure 5 Appendix Figure 6 respectively, And the relationship table between the experimental conditions and "cell density after 24h, cell viability after 24h, expression level after 24h" is as follows: Table 2

[0037] From the data in Table 2, it can be seen that under the same conditions, the higher the total cell number, the higher the protein expression level and the higher the electroporation efficiency. Considering the cell viability and expression level comprehensively, it is considered that the cell viability and expression level of the 5th group are both within a relatively appropriate range.

[0038] Example 3: On the basis of adopting the above "specific implementation process", select "cell number: 1.5×10 7 , plasmid amount: 40 μg, pulse voltages are respectively: 240V, 270V, 300V, 330V, pulse time: 25 ms" as the experimental conditions. After transfection with the expression plasmid containing the target gene, the transfection efficiency and cell viability after 24h are detected. The detection graphs of the expression level, cell density, and cell viability after 24h of electroporation transfection are shown in Appendix Figure 7 , Appendix Figure 8 , and Appendix Figure 9 respectively, And the relationship table between the experimental conditions and "cell density after 24h, cell viability after 24h, expression level after 24h" is as follows: Table 3

[0039] From the data in Table 3, it can be seen that under the same conditions, when the voltage is 270V, the protein expression level is the highest and the electroporation efficiency is the highest.

[0040] Example 4: On the basis of adopting the above "specific implementation process", select "cell number: 1.5×10 7 , plasmid amounts are respectively: 25 μg, 30 μg, 40 μg, 50 μg, 60 μg, pulse voltage: 270V, pulse time: 25 ms" as the experimental conditions. After transfection with the expression plasmid containing the target gene, the transfection efficiency and cell viability after 24h are detected. The detection graphs of the expression level, cell density, and cell viability after 24h of electroporation transfection are shown in Appendix Figure 10 , Appendix Figure 11 , and Appendix Figure 12 respectively, And the relationship table between the experimental conditions and "cell density after 24h, cell viability after 24h, expression level after 24h" is as follows: Table 4

[0041] As can be seen from the data in Table 4, under the same conditions, the higher the concentration of the plasmid, the higher the protein expression level and the higher the electroporation efficiency. After 24 hours of cell transfection, the cell viability was above 60%, and the expression level was between 3 - 11 mg / L. Among them, when the plasmid amount was 60 μg, the highest expression level was 10.17 mg / L.

[0042] From the comprehensive data of Table 1, Table 2, Table 3 and Table 4, it can be seen that under the conditions of a voltage of 270 V, a pulse time of 25 ms, an exponentially decaying wave, in a 4 mm electroporation cuvette, a plasmid amount of 60 μg, and a cell volume of 1.5E7, the transfection effect is the best.

[0043] The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it. However, it should not be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for electroporation transfection of Chinese hamster ovary cells, characterized in that: The steps include: Step 1: Cell preparation: Thaw CHO cells and transfer them to CD303 medium containing L-Glutamine for suspension growth. Subculture the cells for three generations. Step 2: Cell treatment: Add fresh complete culture medium for cell culture into the cell culture flask and preheat it. Take 0.5 to 2 × 10 cells. 7 Place the CHO cells in a centrifuge tube, discard the supernatant after centrifugation, add PBS solvent, discard the supernatant after centrifugation, add PBS solvent to each reaction, and pipette evenly to form a cell suspension; Step 3: Add 25μg~60μg of plasmid to a centrifuge tube, add PBS solvent and mix gently, then add the plasmid to the cell suspension; aspirate the cell and plasmid mixture, and use electroporation transfection to introduce the recombinant plasmid into Chinese hamster ovary CHO cells, and optimize the amount of transfected cells; perform electroporation transfection test on 0.5E7~2E7 cells; perform electroporation transfection test on 25μg~60μg of plasmid; Step 4: Perform cell culture on the samples after electroporation, and then perform cell counting and expression level detection after culture.

2. The method for electroporation transfection of Chinese hamster ovary cells according to claim 1, characterized in that: In step 1, when preparing cells, the CHO cells need to be placed in a 37°C water bath for 2 to 3 minutes to completely lyse the cells.

3. The method for electroporation transfection of Chinese hamster ovary cells according to claim 1, characterized in that: In step 1, 4 mM L-Glutamine was added to the CD303 culture medium, and a suspension growth condition of 5% CO2 concentration and 37°C was provided.

4. The method for electroporation transfection of Chinese hamster ovary cells according to claim 1, characterized in that: In step 2, the cell culture flask is a T-25 cell culture flask, and 7 mL of complete culture medium for fresh cell passage is added therein, wherein the complete culture medium contains 4 mM Glutamine and is preheated at 37°C.

5. The method for electroporation transfection of Chinese hamster ovary cells according to claim 1, characterized in that: In step 2, the centrifugal treatment is performed at a speed of 1000 rpm for 5 min; the amount of PBS solvent added is 0.3 mL to 0.5 mL, and the cell suspension is placed at room temperature for less than or equal to 30 min.

6. The method for electroporation transfection of Chinese hamster ovary cells according to claim 1, characterized in that: In step three, the electroporation conditions are: pulse voltage 240-330V, pulse time 20-30ms, and pulse number not less than one.

7. The method for electroporation transfection of Chinese hamster ovary cells according to claim 6, characterized in that: The pulse voltage is 270V, and the pulse time is 25ms.

8. The method for electroporation transfection of Chinese hamster ovary cells according to claim 1, characterized in that: In step three, the amount of PBS solvent added is 0.4 mL to 0.6 mL.

9. The method for electroporation transfection of Chinese hamster ovary cells according to claim 1, characterized in that: In step 4, the cell culture time is not less than 24 hours.

10. The method for electroporation transfection of Chinese hamster ovary cells according to claim 1, characterized in that: The amount of the plasmid was 60 μg, and the cell volume was 1.5E7.

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