A method for improving the transient transfection efficiency of HEK293 suspension cells
By adding CaCl2 reagent to the HEK293 cell culture medium and optimizing the feed, combined with the liposome reagent PEI for gene delivery, the problem of low transient transfection efficiency of HEK293 suspension cells was solved, and efficient recombinant protein expression and increased yield were achieved.
Patent Information
- Application Number
- CN202310650964.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-02
- Publication Date
- 2025-10-14
- Estimated Expiration
- 2043-06-02
AI Technical Summary
The existing transient transfection efficiency of HEK293 suspension cells is low, which affects the consistency and yield of recombinant protein expression. In addition, traditional methods have safety risks and operational complexity.
CaCl2 reagent was added to the HEK293 cell culture medium, and the cells were fed after transfection. The culture conditions were optimized to improve the transfection efficiency, and the liposome reagent PEI was used for gene delivery.
The expression level and transfection efficiency of exogenous proteins in HEK293 cells were significantly improved, the experimental cycle was shortened, the cost was reduced, and the yield and consistency of recombinant proteins were improved.
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Figure CN116694682B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of improving HEK293 suspension cell transient transfection technology, in particular to a method for improving the efficiency of HEK293 suspension cell transient transfection. BACKGROUND
[0002] Since human embryonic kidney cells were established in the 1970s, HEK293 cells have been widely used for the production of recombinant proteins, virus-like particles and viral vectors due to their high transfection efficiency, rapid growth and high similarity of expressed proteins to natural human proteins. They are widely used in the fields of vaccines, gene therapy, protein therapeutic drugs, diagnosis and scientific research. The expression level of HEK293 cell transient expression of recombinant proteins is usually in the range of milligrams to grams, which varies greatly, so it is very important to deeply study the key factors affecting HEK293 transient expression.
[0003] Cells in suspension culture can be stored in culture bottles without tissue culture treatment, but as the culture volume and surface area increase, sufficient gas exchange will be hindered, and the culture medium needs to be stirred. This stirring is usually achieved by a magnetic stirrer or an oscillating conical flask bottle. Enzymatic digestion or mechanical dissociation is not required, and it is easier to pass and easier to achieve large-scale culture.
[0004] Cell transfection methods can be roughly divided into biological methods, chemical methods and physical methods. Biological methods use viral vectors to achieve gene transfer, but there are safety problems. Chemical methods, such as liposome reagent PEI, have high gene transfer ability in many cell lines, while showing low cytotoxicity, and have the advantages of safety, large-scale production and delivery of large gene fragments. Physical methods can use a variety of physical tools to deliver genetic material to achieve batch transfection or single-cell transfection, such as acoustic penetration and electroporation.
[0005] Recombinant proteins are widely used in scientific research, drug development, disease diagnosis and treatment, and have very important commercial value. Transfection is divided into transient transfection and stable transfection, and the traditional mammalian cell expression system is a stable strain expression system that integrates foreign genes into the host cell chromosome for stable expression. This process usually takes several months and requires a lot of manpower. The number of transient transfection foreign DNA in the cell usually decreases with cell division, and the expression product of the gene can be obtained in a few days to ten days, and its versatility is strong, so the protein transient expression technology is widely used.
[0006] Appropriate formula of trace metal elements, including selenium, zinc, copper and calcium, etc., in the culture medium to optimize its other ingredients is conducive to scalability, improve batch-to-batch performance, improve the consistency of the maximum viable cell density, cell viability, and improve the transient efficiency, wherein Ca2+ has a significant impact on the problem of cell clumping properties, cell growth differentiation and product yield, and therefore we propose a method for improving the transient transfection efficiency of HEK293 suspension cells. SUMMARY
[0007] The purpose of the present application is to provide a method for improving the transient transfection efficiency of HEK293 suspension cells, which solves the existing problems.
[0008] To achieve the above object, the present application provides the following technical solution: a method for improving the transient transfection efficiency of HEK293 suspension cells, comprising the following steps:
[0009] (1) Strain activation
[0010] The E. coli strain preserved in the -80℃ low temperature refrigerator is inoculated on the solid culture medium slope which has been sterilized, and is cultured at 37℃ overnight to activate the strain;
[0011] (2) E. coli 5α shake flask culture
[0012] The colonies on the slope are picked up and inoculated into the liquid medium, and are cultured at 220rpm and 37℃ overnight;
[0013] (3) Extraction of plasmid
[0014] The E. coli in step (2) is centrifuged, and the plasmid is extracted using an endotoxin-free plasmid extraction kit. The extracted plasmid is measured for plasmid concentration and purity.
[0015] Preferably, in step (1), the solid culture medium is composed of Tryptone 10g / L, Yeast Extract 5g / L, NaCl 10g / L, and agar 20g / L, which is sterilized at 121℃ for 30min in advance.
[0016] Preferably, in step (1), the liquid culture medium is composed of Tryptone 10g / L, Yeast Extract 5g / L, and NaCl 10g / L, which is sterilized at 121℃ for 30min in advance.
[0017] The method for transient transfection of HEK293 cells comprises the following steps:
[0018] (1) First, the lipid complex PEI is mixed with the plasmid extracted above in a Ca 2+Incubate in the medium for 25-30 min to obtain PEI / DNA complex;
[0019] (2) Slowly add the transfection complex PEI / DNA into HEK293 cells, and culture under the condition of 120 rpm, 37 DEG C, and 5% CO2;
[0020] (3) After 24 h of cell transfection, perform feeding: 5% FM01A+0.5% FM01B, so as to ensure that the sugar content is 8 g / L, and the residual sugar content is not less than 2 g / L;
[0021] Preferably, the incubation medium in the step (1) is Shanghai Youyi medium CD01 containing 10 mg / L CaCl2.
[0022] Preferably, the cell density in the step (1) is 4*106 cells / mL.
[0023] Preferably, the DNA content in the step (1) is 1.5 mg / L, and the PEI / DNA ratio is 3.
[0024] Two plasmids are used in the experiment, EGFP plasmid is used as the target plasmid first, and then PD-1 plasmid is used for verification.
[0025] Compared with the prior art, the present application has the following beneficial effects:
[0026] The HEK293 suspension cells used in the experiment have the advantages of fast growth speed, strong cell activity, easy control, short experimental period, simple operation and good experimental repeatability.
[0027] The present application provides a HEK293 cell transient expression system transfection method, in the present application, CaCl2 reagent is additionally added in the culture medium, and feeding is performed after 24 h of cell transfection, so as to improve the expression level of HEK293 cell exogenous protein, so that the target gene can be efficiently expressed, and the experimental cost is saved. BRIEF DESCRIPTION OF DRAWINGS
[0028] Figure 1 : Electrophoretogram of EGFP protein expression;
[0029] Figure 2 A-C: Flow cytometry results of cells after 48 h of transient culture in the medium containing different concentrations of Ca 2+ The highest efficiency is 67.9%; Figure 2 D: Transfection efficiency line graph of cells after 48 h of transient culture in the medium containing different Ca 2+
[0030] Figure 3 A: Cells transiently transfected with EGFP plasmid were cultured in the presence of different concentrations of Ca 2+ The cell viability and density of transiently cultured cells in the culture medium for 5 days; Figure 3 B. Cells transiently transfected with PD1 plasmid were cultured in the presence of different concentrations of Ca 2+ The cell viability and density of transiently cultured cells in the culture medium for 5 days;
[0031] Figure 4 : After transient transfection of PD1 plasmid into cells, the cells were harvested after 5 days of culture and incubated with different concentrations of Ca 2+ The yield of transient culture products in the culture medium was the highest for PD1, with a yield of 87 mg / L; No. 1: the culture medium contained 1 mg / L CaCl2; No. 2: the culture medium contained 2 mg / L CaCl2; No. 3: the culture medium contained 5 mg / L CaCl2; No. 4: the culture medium contained 10 mg / L CaCl2; No. 5: the culture medium contained 15 mg / L CaCl2;
[0032] Figure 5 : The relative expression level of EGFP after transient transfection in culture medium containing 10 mg / mL CaCl2 and culture medium without CaCl2 at different times was investigated by qPCR (Real-time Quantitative PCR), i.e., real-time fluorescence quantitative polymerase chain reaction. The expression level of the group transiently transfected in culture medium without CaCl2 for 2 hours was used as the control group. DETAILED DESCRIPTION
[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.
[0034] See also Figures 1-5 A method for improving the transient transfection efficiency of HEK293 suspension cells comprises the following steps:
[0035] (1) Bacteria activation
[0036] The Escherichia coli strain stored in a -80°C freezer was selected and inoculated onto a sterile solid culture medium slant, and cultured overnight at 37°C for strain activation. In step (1), the solid culture medium was composed of: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, and agar 20 g / L, and was pre-sterilized at 121°C for 30 min.
[0037] (2) Shake flask culture of Escherichia coli 5α
[0038] The colonies are picked from the slope and inoculated into liquid medium, and the liquid medium is cultured overnight at 220 rpm and 37 DEG C, and the composition of the liquid medium in step (1) is as follows: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, and the liquid medium is sterilized at 121 DEG C for 30 min in advance;
[0039] (3) Extraction of plasmid
[0040] The E. coli in step (2) is centrifuged, and the plasmid is extracted by using an endotoxin-free plasmid extraction kit, and the extracted plasmid is measured for plasmid concentration and purity.
[0041] The HEK293 cell transient transfection method comprises the following steps:
[0042] (1) The lipid complex PEI is incubated with the plasmid extracted from the above for 25-30 min in a culture medium containing Ca 2+ to obtain a PEI / DNA complex;
[0043] In step (1), the incubation culture medium is a Shanghai Youyi culture medium CD01 containing 10 mg / L of CaCl2, the cell density in step (1) is 4 x 106 cells / mL, the DNA content in step (1) is 1.5 mg / L, and the PEI / DNA ratio is 3.
[0044] (2) The transfection complex PEI / DNA is slowly added to the HEK293 cells, and the cells are cultured in suspension at 120 rpm, 37 DEG C and 5% CO2;
[0045] (3) After the cells are transfected for 24 h, the feeding is performed: 5% FM01A + 0.5% FM01B, so that the sugar content is 8 g / L, and the residual sugar content is not less than 2 g / L.
[0046] The following are used in the embodiments of the application:
[0047] The strain name is E. coli DH5a, which is preserved by the State Key Laboratory of Biochemical Engineering, East China University of Science and Technology.
[0048] The cell name is HEK293 (human embryonic kidney 293) cell, which is preserved by the State Key Laboratory of Biochemical Engineering, East China University of Science and Technology.
[0049] The flow cytometry analysis method used in the embodiments of the application is as follows:
[0050] After the EGFP plasmid is transiently introduced into the HEK293 cells for 48 h, 1 x 106 cells are taken, and the cells are resuspended in 1 mL of PBS, and the cell concentration is adjusted to 1 x 106 cells / mL. 6The cells were placed in an EP tube, centrifuged at 1000 rpm for 10 min, the supernatant was poured out, then washed twice with PBS, passed through a 300-mesh sieve, and then detected using the FITC channel
[0051] The names, specifications, and manufacturers of the main reagents used in the present application are as follows:
[0052]
[0053]
[0054] The names, specifications, and manufacturers of the main instruments used in the present application are as follows:
[0055]
[0056]
[0057] Examples
[0058] (1) Plasmid extraction
[0059] An E. coli strain preserved in a -80℃ low-temperature refrigerator was inoculated on a solid culture medium slope that had been sterilized, and was cultured at 37℃ overnight to activate the strain;
[0060] The solid culture medium was composed of Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, and agar 20 g / L, and was sterilized at 121℃ for 30 min in advance.
[0061] A colony was picked from the slope and inoculated in 200 mL of liquid culture medium, and was cultured at 220 rpm and 37℃ overnight.
[0062] The liquid culture medium was composed of Tryptone 10 g / L, Yeast Extract 5 g / L, and NaCl 10 g / L, and was sterilized at 121℃ for 30 min in advance.
[0063] The E. coli was collected and centrifuged, and a non-endotoxin plasmid extraction kit was used to extract the plasmid according to the steps in the instructions.
[0064] (2) Suspension culture of HEK293 cells
[0065] The cryopreservation tube was taken out from the designated position in the liquid nitrogen storage box, quickly placed in a 37℃ water bath, and gently shaken to thaw the tube within 1 min, and the cells were transferred to a 125 mL shake flask with a culture volume of 30 mL, and were cultured at 37℃ and 120 rpm.
[0066] When the cell density grows to 4 x 10 6 When the cell density grows to 4 x 10
[0067] (3) Transient transfection of HEK293 cells
[0068] The lipid complex PEI was diluted in the medium containing different concentrations of Ca 2+ (1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 15 mg / L) and then the PEI solution was slowly added to the DNA solution and incubated for 25-30 min to obtain the PEI / DNA complex;
[0069] The above PEI / DNA complex was immediately added to the cells for transient transfection;
[0070] (4) Detection of transient transfection efficiency
[0071] After 48 h of transient transfection of EGFP plasmid into HEK293 cells, 1 x 10 6 cells were placed in a sterile EP tube, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded after resuspension with PBS and centrifugation at 1000 rpm for 10 min. The cells were washed once more, passed through a 300-mesh sieve, and then detected. The detection instrument was a Beckman Coulter flow cytometer.
[0072] (5) Detection of PD-1 production
[0073] After centrifugation at 1000 rpm for 10 min, 300 μL of supernatant was taken and the content of PD-1 protein was detected using Roche CedexBioHT biochemical analyzer.
[0074] As can be seen from the results, after the suspension HEK293 cell culture density reached 4 x 10 6 After 48 h of transient transfection of EGFP plasmid, the transient transfection efficiency was determined by flow cytometry. The highest transient transfection efficiency was 67.9% in the culture medium containing 10 mg / L CaCl2. On this basis, PD-1 plasmid was used for result verification. The results showed that the highest product yield was 87 mg / mL in the culture medium containing 10 mg / L Ca 2+
[0075] Ca 2+ has a certain promoting effect on cell growth and transient transfection efficiency, but excessive Ca 2+ The addition of CaCl2 can cause cell aggregation to precipitate, further reduce cell filtration and affect the transfection efficiency. The transfection efficiency in the culture medium containing 10 mg / L CaCl2 is 67.9%, which is nearly twice as high as the original; on this basis, the PD1 plasmid is used for result verification, and the results show that the culture medium containing 10 mg / L CaCl2 is used for incubation and transfection, and the highest product yield is 87 mg / mL, which is 7.25 times higher than the original 12 mg / L. 2+ The culture medium containing 10 mg / L CaCl2 is used for incubation and transfection, and the highest product yield is 87 mg / mL, which is 7.25 times higher than the original 12 mg / L.
[0076] The transcription level of the final plasmid EGFP is obtained by qPCR technology, and the expression level of the group without CaCl2 in the culture medium for 2 h is used as the control group. It can be known that with the increase of transfection time, the content of cells expressing EGFP protein is more and more, and the addition of 10 mg / L CaCl2 solution has a promoting effect on the transcription and translation after transfection.
[0077] The above is only an example of the embodiment of the present application, and it should be pointed out that for ordinary skilled in the art, without departing from the technical principles of the present application, a number of improvements and modifications can be made, and these improvements and modifications should be considered as the protection scope of the present application.
Claims
1. A method for transiently transferring a plasmid into HEK293 cells to express a protein, characterized in that: The following steps are involved: (1) Bacteria activation The E. coli strain stored in a -80℃ freezer was selected and inoculated onto a sterile solid culture medium slant, and cultured overnight at 37℃ for strain activation. (2) Shake flask culture of Escherichia coli DH5α Pick colonies from the slant and inoculate into liquid culture medium, culture at 220 rpm and 37°C overnight; (3) Plasmid extraction The E. coli in step (2) was centrifuged and the plasmid was extracted using an endotoxin-free plasmid extraction kit. The concentration and purity of the extracted plasmid were measured. The transient transfection method of HEK293 cells includes the following steps: (a) First, the lipid complex PEI and the plasmid extracted from the above were mixed in a medium containing Incubate in the culture medium for 25-30 minutes to obtain PEI / DNA complex; (b) The PEI / DNA transfection complex was slowly added dropwise to HEK293 cells and cultured in suspension at 120 rpm, 37°C, and 5% CO2. (c) 24 h after cell transfection, feed the cells with 5% FM01A + 0.5% FM01B to ensure a sugar content of 8 g / L and a residual sugar content of no less than 2 g / L. The incubation medium in step (a) is Shanghai Youyi medium CD01 containing 10 mg / L CaCl2; The cell density during transfection in step (b) was 4×10 6 cells / mL; During transfection in step (a), the DNA content was 1.5 mg / L and the PEI / DNA ratio was 3.
2. The method for transiently transferring a plasmid into HEK293 cells for protein expression according to claim 1, wherein: In step (1), the solid culture medium is composed of: 10 g / L Tryptone, 5 g / L Yeast Extract, 10 g / L NaCl, and 20 g / L agar, and is pre-sterilized at 121°C for 30 min.
3. The method for transiently transferring a plasmid into HEK293 cells to express a protein according to claim 1, characterized in that: In step (2), the composition of the liquid culture medium is: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, and is pre-sterilized at 121°C for 30 min.