Method for improving PEI transfection efficiency of Chinese hamster ovary cells

Abstract

The invention discloses a method for improving PEI transfection efficiency of Chinese hamster ovary cells. The method comprises the following steps: S1, inoculating CHO cells into a culture medium for cell culture; s2, adjusting the cell density to (1.5-3) * 10 < 6 > cells / mL one day before transfection; s3, adjusting the cell density to (5-7) * 10 < 6 > cells / mL on the transfection day; s4, 25 to 375 umol / L of calcium salt is added; s5, adding a PEI and target plasmid compound and dropwise adding the compound into the cells; s6, after transfection is conducted for 24 h, 1.5 mmol / L to 3 mmol / L of sodium valproate or sodium butyrate is supplemented. According to the method, the transient transfection efficiency of the CHO cells can be remarkably improved, so that the expression quantity of protein is improved, and the cell culture cost is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a method for improving the PEI transfection efficiency of Chinese hamster ovary cells.

Background Art

[0002] Chinese Hamster Ovary (CHO) cells are the most widely used host cells for the production of antibodies and protein drugs. More than 70% of the commercially available therapeutic protein drugs are produced by CHO cells. Compared with other mammalian cell expression systems, CHO cells have the advantages of post-translational modification similar to that of humans, clear historical background, rapid growth, and high expression level.

[0003] In order to obtain antibodies or proteins in a short time, transient transfection technology is widely used, including physical methods (such as electroporation), chemical methods, and biological methods (such as Sendai virus). Due to its convenience and low cost, the chemical method has become the most important transient transfection method. Chemical transfection methods mainly include calcium phosphate co-precipitation method and cationic polymers. The calcium phosphate co-precipitation method on CHO cells has strong toxicity and low transfection efficiency, and is basically not used.

[0004] Polyethyleneimine (PEI) is a widely studied cationic polymer that can enter cells by interacting with cell surface proteoglycans and bring exogenous plasmids into cells (as shown in Figure 1 ). PEI is divided into linear and branched forms. The transfection efficiency of PEI on CHO cells is relatively low, usually only about 40%.

[0005] In the prior art, Patent 202411879537.X discloses a transfection enhancement method for improving the transient transfection efficiency of plasmids, which can rapidly increase the transient transfection expression level of MATE2-K by optimizing the transfection steps and the content of lithium acetate. However, since this transfection is based on Lipofectamine TM 3000, and Lipofectamine TM 3000 is a commercial liposome transfection reagent developed by Thermo Fisher Scientific in the United States, with a high price, which limits the application of this method.

[0006] Therefore, it is necessary to provide a new method for improving the PEI transfection efficiency of Chinese hamster ovary cells to solve the above technical problems.

Summary of the Invention

[0007] The main object of the present invention is to provide a method for improving the PEI transfection efficiency of Chinese hamster ovary cells, which can significantly improve the transient transfection efficiency of CHO cells, thereby increasing the protein expression level.

[0008] The present invention achieves the above object through the following technical solutions: A method for improving the transfection efficiency of PEI in Chinese hamster ovary cells, which comprises the following steps:

[0009] S1. Inoculate CHO cells into a culture medium for cell culture;

[0010] S2. One day before transfection, adjust the cell density to (1.5 - 3)×10 6 cells / mL;

[0011] S3. On the day of transfection, adjust the cell density to (5 - 7)×10 6 cells / mL;

[0012] S4. Add 25 - 375 umol / L of calcium salt;

[0013] S5. Add the PEI and target plasmid DNA complex dropwise to the above cells;

[0014] S6. At 24 h after transfection, supplement 1.5 - 3 mmol / L of sodium valproate or sodium butyrate.

[0015] The present invention has the following effects by adding calcium ions during the incubation of the PEI - DNA complex:

[0016] (1) Enhance the stability of the complex and cell uptake.

[0017] Ca 2 ²⁺, as a divalent cation, can neutralize the negative charge of the DNA phosphate backbone, promote the closer condensation of PEI (positively charged) with DNA, form smaller - sized and more stable nanoparticles (100 - 200 nm), reduce aggregation, and improve the homogeneity of the complex;

[0018] Ca 2 ²⁺ enhances macropinocytosis or clathrin - mediated endocytosis by activating calcium - dependent signaling pathways (such as calmodulin) on the cell membrane, and improves the efficiency of the complex entering the cell.

[0019] (2) Assist endosomal escape.

[0020] The "proton sponge effect" of PEI depends on endosomal acidification. Ca 2 ²⁺ binds to endosomal membrane phospholipids (such as phosphatidylserine), increases membrane permeability, promotes the release of the PEI - DNA complex into the cytoplasm, and then synergistically disrupts the stability of the endosomal membrane.

[0021] Figure 5 shows the effect of calcium ion addition on the formation of the PEI and DNA complex. From Figure 5It can be clearly seen that the addition of calcium ions can make the complex of PEI and DNA more compact, improve the ability of the PEI-DNA complex to enter cells, and thus improve the transfer efficiency and expression level.

[0022] In the expression stage of the PEI-DNA complex of the present invention, sodium butyrate or sodium valproate is added as an inhibitor of histone deacetylase, and specifically has the following effects:

[0023] (1) Epigenetic regulation.

[0024] Sodium butyrate and sodium valproate (VPA) can inhibit histone deacetylase HDAC, resulting in an increase in histone acetylation level, an open chromatin structure, and making the exogenous plasmid DNA more accessible to the transcriptional machinery;

[0025] Sodium valproate or sodium butyrate, as an inhibitor of histone deacetylase, can up-regulate anti-apoptotic genes (such as Bcl-2), slow down the rate of cell entry into apoptosis, and extend the protein synthesis window period;

[0026] Sodium valproate or sodium butyrate arrests CHO cells in the G1 phase. At this time, the cells have active metabolism and strong transcription, which is beneficial to the high-efficiency expression of exogenous genes. By strictly controlling the concentration (1.5 - 3 mmol / L) and treatment time (added 24 hours after transfection), excessive inhibition of cell growth is avoided;

[0027] Sodium valproate or sodium butyrate, as an inhibitor of histone deacetylase, can up-regulate the expression of molecular chaperones (such as BiP / Grp78), assist the correct folding of recombinant proteins, reduce aggregate formation, which is particularly important for the expression of secreted antibodies (such as PD-L1mAb).

[0028] Preferably, the cell density of the CHO cells inoculated into the culture medium in step S1 is 0.5×10 6 cells / mL.

[0029] Preferably, in step S3, centrifuge at 350 g for 5 min to discard the cell supernatant, and then resuspend with fresh culture medium, and adjust the cell density to (5 - 7)×10 6 cells / mL.

[0030] Preferably, in step S3, directly adjust the cell density to (5 - 7)×10 6 cells / mL with fresh culture medium.

[0031] Preferably, the fresh culture medium is produced by Shanghai Youyi Biotechnology Co., Ltd., and the product number is TransCD05.

[0032] Preferably, the calcium salt is calcium chloride or calcium sulfate.

[0033] Preferably, the target plasmid in the PEI and target plasmid complex is an eGFP plasmid or a PD-L1 monoclonal antibody plasmid.

[0034] Preferably, the ratio of PEI to the target plasmid in the PEI and target plasmid complex is 5:1.

[0035] Preferably, in step S1, the environmental temperature for cell culture is 37 °C, and the culture method is suspension culture.

[0036] Compared with the prior art, the beneficial effects of the method for improving the transfection efficiency of PEI in Chinese hamster ovary cells of the present invention are as follows: The addition of calcium ions during the incubation of PEI and the target plasmid DNA and the addition of sodium butyrate or sodium valproate during the expression in the present invention significantly improve the transient transfection efficiency of CHO cells and increase the protein expression level. Specifically:

[0037] (1) By adding calcium salts during the cell culture process to provide calcium ions, the addition of calcium ions can effectively improve the transfection efficiency of CHO cells, obtain more protein expression, and thus reduce production costs; however, the addition concentration of calcium ions needs to be strictly controlled. Excessive addition may cause cytotoxicity. The addition of an appropriate concentration of calcium ions can make the complex of PEI and DNA tighter, improve the ability of the PEI and DNA complex to enter cells, and thus improve the transfection efficiency and expression level;

[0038] (2) By adding sodium valproate or sodium butyrate during the transfection process, the protein expression is further promoted. Sodium butyrate and sodium valproate can promote gene transcription, thereby increasing the synthesis rate of the target protein.

Explanation of the Drawings

[0039] Figure 1 is the schematic diagram of PEI transfection in the prior art;

[0040] Figure 2 is the flow cytometry diagram of Examples 1-5 and Comparative Example 1 of the present invention. Among them, (A) 1.0 mg / L CaCl2; (B) 2.0 mg / L CaCl2; (C) 5.0 mg / L CaCl2; (D) 10.0 mg / L CaCl2; (E) 15.0 mg / L CaCl2; (F) 0.0 mg / L CaCl2;

[0041] Figure 3 is the flow cytometry diagram of Examples 6-10 and Comparative Example 2 of the present invention. Among them, (A) is the yield of anti-PD-L1 antibody; (B) is the SDS-PAGE electrophoresis diagram of PD-1 antibody; (C) is the cell density; (D) is the cell viability;

[0042] Figure 4This is the detection result diagram of Examples 11 - 14 of the present invention and Comparative Example 3, where (A) is the transfection efficiency; (B) is the SDS-Page diagram of protein expression.

[0043] Figure 5 It is the mechanism diagram of the influence of Ca ions on transfection efficiency, where (A) and (B) are transmission electron microscope images of 5.0 mg / L CaCl2; (C) is the transmission electron microscope image without adding calcium ions.

Specific Embodiments

[0044] Examples 1 - 5: A method for improving the PEI transfection efficiency of Chinese hamster ovary cells, which includes the following steps:

[0045] S1. Inoculate CHO cells into the culture medium for cell culture. The culture temperature of CHO cells is 37°C, and the culture method is suspension culture.

[0046] The specific culture medium is Trans CD03, produced by Shanghai Youyi Biotechnology Co., Ltd., with the product number YYCD03.001. The culture method is shake flask culture. The shake flask is purchased from Zhejiang Baidi Technology, with the product number FL125, and the liquid loading volume is 30 mL.

[0047] S2. One day before transfection, adjust the cell density to 2×10 6 cells / mL.

[0048] S3. On the day of transfection, centrifuge at 350 g for 5 min to remove the cell supernatant, and then resuspend the cells with Trans CD03 culture medium to adjust the cell density to (5 - 7)×10 6 cells / mL. This step precipitates the cells to the bottom of the shake flask by low-speed centrifugation (350 g) for 5 min, and at the same time removes components such as serum, antibiotics or other components that may interfere with the transfection reagent in the original culture medium, ensuring that the efficiency of the subsequent transfection reagent is not affected by the components of the original culture medium. By resuspending the cells with Trans CD03 culture medium, the cell state can be optimized and the transfection efficiency can be improved, reducing the interference with the formation of transfection complexes.

[0049] Or directly adjust the cell density to (5 - 7)×10 6 cells / mL with fresh culture medium.

[0050] The adjusted cell density in this step is 6×10 6 cells / mL.

[0051] S4. Add 1 - 15 mg / L calcium chloride.

[0052] Calcium chloride was purchased from Aladdin Biology, with the product number C290953 and a purity of ≥99.99%. Before adding it to the medium, a stock solution of 1500 mg / L was prepared first.

[0053] In order to verify the effect of calcium chloride (CaCl2) on transfection efficiency, multiple examples were set up for experimental verification, and the situations of each example are shown in Table 1.

[0054] Table 1 Addition parameter table of calcium chloride in Examples 1 - 5 and Comparative Example 1

[0055] <![CDATA[CaCl2 addition amount (mg / L)]]> Example 1 1 Example 2 2 Example 3 5 Example 4 10 Example 5 15 Comparative Example 1 0

[0056] S5. Add the PEI and eGFP plasmid complex dropwise to the above cells. The ratio of PEI:eGFP plasmid in the PEI and eGFP plasmid complex is 5:1.

[0057] S6. At 24 h after transfection, supplement 2 mmol / L of sodium valproate (VPA) or sodium butyrate.

[0058] Unless otherwise specified, the reagents and instruments used in the above examples are all conventional selections in the art. For the experimental methods without specific conditions noted in the examples, they are usually carried out under conventional conditions, such as the conditions described in the literature, books, or the methods recommended by the kit manufacturers. The unit mM represents mmol / L, and M represents mol / L.

[0059] Materials used in the experiment:

[0060] CHO cells (Chinese hamster ovary cells) were obtained by Suzhou Youyi Biotechnology Co., Ltd. through commercial channels.

[0061] The medium was provided by Shanghai Youyi Biotechnology Co., Ltd., and it is a medium that can culture CHO cells.

[0062] The cells were cultured in a 125 mL shake flask with an air-permeable cap (Zhejiang Baidi Technology, product number FL125). The shake flask was cultured in a shaker at 37°C and 5% CO2 for 2 days, and the shaker speed was 120 rpm - 130 rpm. On the second day, samples were taken to detect the transfection efficiency.

[0063] The specific statistical test method is as follows:

[0064] Determination of transfection efficiency: Turn on the flow cytometer. First, place the untransfected pre-treated cells on the machine. Set the channel FITC in the Cytexpert software, select the dot plot, and aspirate 10,000 cells for detection under medium-speed conditions. Then take out the untransfected cells and circle the cells with good growth status as the negative control. After pre-treating the cells transfected with the eGFP plasmid under different conditions, place them into the machine in turn for the detection of transient transfection efficiency and record.

[0065] The test results of Examples 1 to 5 and Comparative Example 1 are as follows: Figure 2 As shown, Figure 2 The transfection efficiency of Examples 1 to 5 and Comparative Example 1 under the corresponding CaCl2 addition conditions is shown in Table 2.

[0066] Table 2 Transfection efficiency of Examples 1 to 5 and Comparative Example 1

[0067] <![CDATA[CaCl2 addition amount (mg / L)]]> Transfection efficiency (%) Example 1 1 53.50 Example 2 2 59.32 Example 3 5 54.53 Example 4 10 66.73 Example 5 15 63.14 Comparative Example 1 0 44.42

[0068] according to Figure 2 The results shown in A to F are as follows:

[0069] (1) The addition of CaCl2 can significantly improve the transfection efficiency of CHO cells;

[0070] (2) When the addition amount was within the range of 1 to 10 mg / L, the transfection efficiency increased with the increase of CaCl2 addition amount. The addition effect of 15 mg / L CaCl2 was not as good as that of 10 mg / L CaCl2.

[0071] Embodiments 6 to 10: A method for improving the PEI transfection efficiency of Chinese hamster ovary cells, comprising the following steps:

[0072] S1. CHO cells were inoculated into culture medium for cell culture. The CHO cell culture temperature was 37° C. and the culture method was suspension culture.

[0073] The cell culture medium was Trans CD03, produced by Shanghai Youyi Biotechnology Co., Ltd., with the product number YYCD03.001. The culture method was shake flask culture, purchased from Zhejiang Biotech, with the product number FL125, and the liquid volume was 30 mL.

[0074] S2. The day before transfection, adjust the cell density to 2×10 6 cells / mL.

[0075] S3. On the day of transfection, centrifuge at 350g for 5 minutes to remove the cell supernatant, and then resuspend the cells in Trans CD03 medium to adjust the cell density to 6×10 6 cells / mL.

[0076] S4. Add 1-15 mg / L calcium chloride.

[0077] Calcium chloride was purchased from Aladdin Biotechnology, catalog number C290953, with a purity of ≥99.99%. Before adding it to the culture medium, it was prepared into a stock solution of 1500 mg / L.

[0078] To verify the effect of calcium chloride on transfection efficiency, multiple examples were set up for experimental verification, and the situations of each example are shown in Table 3.

[0079] Table 3 Addition parameter table of calcium chloride in Examples 6 - 10 and Comparative Example 2

[0080] <![CDATA[CaCl2 addition amount (mg / L)]]> Example 6 1 Example 7 2 Example 8 5 Example 9 10 Example 10 15 Comparative Example 2 0

[0081] S5. Add the complex of PEI and PD-L1 monoclonal antibody plasmid dropwise to the above cells. The ratio of (PEI: PD-L1 monoclonal antibody plasmid) in the complex of PEI and PD-L1 monoclonal antibody plasmid is 5:1.

[0082] S6. At 24 h of transfection, supplement 2 mmol / L of sodium valproate (VPA) or sodium butyrate.

[0083] The cells were cultured in a 125 mL shake flask with an air-permeable cap (Zhejiang Baidi Technology, product number FL125). The shake flask was cultured in a shaker at 37°C and 5% CO2 for 2 days, and the shaker speed was 120 rpm - 130 rpm. On the second day, samples were taken for detection of the expression level.

[0084] The specific statistical test method is as follows:

[0085] Determination of antibody expression level: Measured by HPLC method (Agilent, model 1100): Equilibrate the HPLC system with 0.1 M phosphate buffer at a flow rate of 2 mL / min for 15 min until the baseline is stable, inject 50 μL of sample, and elute at a flow rate of 2 mL / min.

[0086] The detection results of Examples 6 - 10 and Comparative Example 2 are as Figure 3 shown. According to Figure 3 the results shown in A - D below, it can be known that:

[0087] (1) The addition of CaCl2 can significantly increase the transfection protein expression level of CHO cells;

[0088] (2) Within the range of 1 - 10 mg / L of the addition amount, as the addition amount of CaCl2 increases, the transfection efficiency is higher. The addition effect of 15 mg / L of CaCl2 is inferior to that of 10 mg / L of CaCl2;

[0089] (3) High CaCl2 concentration will affect cell viability and density.

[0090] Examples 11 - 14: A method for improving the PEI transfection efficiency of Chinese hamster ovary cells, which includes the following steps:

[0091] S1. Inoculate CHO cells into the culture medium for cell culture. The culture temperature of CHO cells is 37°C, and the culture method is suspension culture.

[0092] The specific cell culture medium is Trans CD03, which is produced by Shanghai Youyi Biotechnology Co., Ltd. with the product number YYCD03.001. The culture method is shake flask culture. The shake flasks are purchased from Zhejiang Baidi Technology with the product number FL125, and the liquid loading volume is 30 mL.

[0093] S2. One day before transfection, adjust the cell density to 2×10 6 cells / mL.

[0094] S3. On the day of transfection, centrifuge at 350 g for 5 min to remove the cell supernatant, and then resuspend the cells with Trans CD03 medium to adjust the cell density to 6×10 6 cells / mL.

[0095] S4. Add calcium chloride, and the addition amount of calcium chloride is shown in Table 4.

[0096] Table 4 Addition parameter table of calcium chloride in Examples 11 - 14 and Comparative Example 3

[0097] <![CDATA[CaCl2 addition amount (mg / L)]]> Example 11 10 Example 12 10 Example 13 10 Example 14 10 Comparative Example 3 0

[0098] S5. Add the PEI and eGFP plasmid complex dropwise to the above cells. The ratio of PEI:eGFP plasmid in the PEI and eGFP plasmid complex is 5:1.

[0099] S6. Add sodium valproate (VPA) at 24 h after transfection, and the addition concentration of sodium valproate is shown in Table 5.

[0100] Table 5 Addition parameter table of sodium valproate in Examples 11 - 14 and Comparative Example 3

[0101] VPA addition amount (mmol / L) Example 11 0 Example 12 1.5 Example 13 3 Example 14 4 Comparative Example 3 0

[0102] The cells are cultured in 125 mL shake flasks with air - permeable lids (Zhejiang Baidi Technology, product number FL125). The shake flasks are cultured in a shaker at 37 °C and 5% CO2 for 2 days, and the shaker speed is 120 rpm - 130 rpm. On the second day, take samples to detect the expression level.

[0103] The specific statistical test methods are as follows:

[0104] Determination of antibody expression level: Measured by HPLC method (Agilent, model 1100): Equilibrate the HPLC system with 0.1 M phosphate buffer at a flow rate of 2 mL / min for 15 min until the baseline is stable, inject 50 μL of the sample, and elute at a flow rate of 2 mL / min.

[0105] The detection results of Examples 11 - 14 and Comparative Example 3 are as Figure 4 shown. According toFigure 4 From the results shown in A to B, the following can be obtained:

[0106] (1) The addition of VPA can significantly improve the transfection efficiency of CHO cells;

[0107] (2) Within the range of 1 - 3 mM of the addition amount, as the addition amount of VPA increases, the transfection efficiency is higher; the addition effect of 4 mmol / L VPA is inferior to that of 3 mmol / L VPA.

[0108] For those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, and these all fall within the protection scope of the present invention.

Claims

1. A method for improving the transfection efficiency of PEI in Chinese hamster ovary cells, characterized in that: It includes the following steps: S1. Inoculate CHO cells into a culture medium for cell culture; S2. One day before transfection, adjust the cell density to (1.5 - 3)×10 6 cells / mL; S3. On the day of transfection, adjust the cell density to (5-7)×10 6 cells / mL; S4. Add 25 - 375 μmol / L of calcium salt; S5. Add the complex of PEI and the target plasmid dropwise to the above cells; S6. At 24 h after transfection, supplement 1.5 - 3 mmol / L of sodium valproate or sodium butyrate.

2. The method for improving the PEI transfection efficiency of Chinese hamster ovary cells according to claim 1, characterized in that: The cell density of the CHO cells inoculated into the culture medium in step S1 is 0.5×10 6 cells / mL.

3. The method for improving the transfection efficiency of PEI in Chinese hamster ovary cells according to claim 1, wherein: In step S3, centrifuge at 350 g for 5 min to discard the cell supernatant, then resuspend with fresh medium and adjust the cell density to (5-7)×10 6 cells / mL.

4. The method for improving the transfection efficiency of PEI in Chinese hamster ovary cells according to claim 1, characterized in that: In step S3, directly adjust the cell density to (5-7)×10 6 cells / mL using fresh culture medium.

5. The method for improving the transfection efficiency of PEI in Chinese hamster ovary cells according to claim 3 or 4, characterized in that: The fresh culture medium is produced by Shanghai Youyi Biotechnology Co., Ltd., with the product number Trans CD05.

6. The method for improving the transfection efficiency of PEI in Chinese hamster ovary cells according to claim 1, characterized in that: The calcium salt is calcium chloride or calcium sulfate.

7. The method for improving the transfection efficiency of PEI in Chinese hamster ovary cells according to claim 1, characterized in that: The target plasmid in the complex of PEI and the target plasmid is eGFP plasmid or PD-L1 monoclonal antibody plasmid.

8. The method for improving the PEI transfection efficiency of Chinese hamster ovary cells according to claim 7, characterized in that: The ratio of PEI to the target plasmid in the complex of PEI and the target plasmid is 5:

1.

9. The method for improving the transfection efficiency of PEI in Chinese hamster ovary cells according to claim 1, characterized in that: In step S1, the environmental temperature for cell culture is 37 °C, and the culture method is suspension culture.

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