CN112824530A

CN112824530A - HEK293F suspension cell high-efficiency electrotransfection method

Info

Publication number: CN112824530A
Application number: CN201911140162.4A
Authority: CN
Inventors: 金宗文; 卫小元; 罗擎颖; 赵江林; 刘宇光; 苏少博
Original assignee: Really Tech Co ltd; Shenzhen Institute of Advanced Technology of CAS
Current assignee: Really Tech Co ltd; Shenzhen Institute of Advanced Technology of CAS
Priority date: 2019-11-20
Filing date: 2019-11-20
Publication date: 2021-05-21

Abstract

The invention provides a high-efficiency electrotransfection method for HEK293F suspension cells, which optimizes the aspects of voltage intensity, pulse frequency, pulse interval duration, electric shock duration and the like during electrotransfection to realize high-efficiency electrotransfection of HEK293F cells cultured in suspension, and can obtain higher transfection efficiency without preparing electrotransfection buffer solution during electrotransfection. The method can be used for transfecting HEK293F cells cultured in suspension by different plasmids to obtain eukaryotic expression of a large number of target proteins (such as protein products of antibodies, enzymes and the like).

Description

HEK293F suspension cell high-efficiency electrotransfection method

Technical Field

The invention belongs to the technical field of transgenosis, and particularly relates to a high-efficiency electrotransfection method for HEK293F suspension cells.

Background

Liposome transfection method is the most used transfection method at present, and liposome wraps DNA to form DNA/liposome complex, and cells transfer DNA into cells by means of endocytosis. However, the quality of DNA has a great influence on the transfection efficiency, and small doses of endotoxin, salt ions, proteins, polysaccharides and the like all influence the formation of complexes, so that the long-term contact of low-quality DNA with cells can cause the increase of cell death rate and reduce the transfection efficiency. Moreover, the liposome on the market is high in price and is not suitable for large-volume transfection. PEI is cheap, but has low transfection efficiency and high cytotoxicity. Calcium phosphate transfected cells require large amounts of DNA and are sensitive to pH changes in the transfection buffer.

In order to solve the problem of low efficiency of the lipofection method, researchers propose a method using electrotransfection. Cell electrotransfection, also called electroporation, is an important method for introducing exogenous macromolecular substances such as DNA, RNA, siRNA, proteins, and small molecules into the interior of cell membranes. Under the applied electric field, the cell membrane has certain permeability, so that charged exogenous substances such as DNA and the like can enter the cell in a manner similar to electrophoresis. Since the cell membrane is composed of a phospholipid bilayer and is similar to an insulator, almost no current flows inside the cytoplasm in an electric field, and DNA adsorbed on the surface of the cell membrane enters the cell by the flow of the cell membrane. When the conditions are proper, the toxicity of the external voltage to the cells is small, and the cells which survive the electrotransfection can proliferate normally.

At present, there are many reports on the optimization of cell electrotransfection conditions, including macrophages, dendritic cells, fibroblasts, CHO cells, SGC7901/ADM and the like. Down rock et al gave the highest transfection efficiency of dendritic cells at a voltage pulse of 500 μ s at 300V. Zygui et al transfected HepG2 and SGC7901/ADM cells by electrotransfection, and both gave transfection efficiencies of about 50%. For HepG2 cells, 20. mu.g of DNA was mixed with 2X 10 DNA in the electrotransfer buffer⁶Mixing the cells, incubating at 4 deg.C for 10min, and applying a voltage of 270V to obtain a mixturePulse is carried out for 20ms, and after electrotransfection, the cells are placed in a DEME high-sugar medium containing 15% FBS at 37 ℃ for 48h to obtain the highest transfection efficiency of 60%.

Electrotransfection is to apply external electric field strength to cell to make cell membrane have micropores, temporarily lose shielding effect, and DNA outside cell membrane can enter cell through micropores. For the same kind of cells, the electric field intensity is too low to form micropores, and the transfection efficiency is low; the electric field intensity is too high, great irreversible damage is generated to cells, and the cell death rate is high. In the existing electrotransfection technology, the optimal electrotransfection conditions are different for different cells; the cell size and the structural components of the cell membrane are different, and the electrotransformation conditions are also different. Therefore, optimal electrotransfection conditions need to be explored for a particular cell. Dendritic cells are the antigen presenting cells with the strongest functions discovered at present, HepG2 cells are important cell models for liver cancer research, and the HepG2 cells and the adherent cells are focus cells in the field of tumor biological treatment; however, when electrotransfection is performed on dendritic cells and HepG2 cells, the cell density is high, so that the threshold value of some cells is difficult to reach. Under the reported optimal electrotransfer conditions of the DC cells and the HepG2 cells, the electrotransfer efficiency is 50-60%, but the problems that an electrotransfer buffer solution is needed during electrotransfer, incubation is carried out at 4 ℃ before electrotransfer, the use amount of nucleic acid is large and the like exist. The HEK293F cell is a cell which is separated from the HEK293 cell and is suitable for suspension cell culture, and can be used for large-scale expression of eukaryotic proteins. As a eukaryotic expression host, eukaryotic proteins which are difficult to express by prokaryotic cells can be expressed, and the proteins can be subjected to post-translational modification.

Aiming at the problems, the specific suspension cell line HEK293F is selected, the voltage intensity, the pulse frequency, the pulse interval duration, the electric shock duration and other aspects during electrotransfection are optimized, the effects of simplicity and convenience in operation, high transfection efficiency, low cytotoxicity and the like are achieved, the method can perform large-volume transfection without pretreatment and the use of electrotransfection buffer solution, and is applied to cell biology, immunology, hematology, cancer research, antibody, new drug research and development and the like.

Disclosure of Invention

In view of this, the present invention aims to provide a method for efficiently electrotransfecting HEK293F suspension cells, which is simple and convenient to operate, has high transfection efficiency and low cytotoxicity.

In order to realize the purpose of the invention, the invention adopts the following technical scheme: an HEK293F suspension cell high-efficiency electrotransfection method, 1) preparing transfection plasmid; 2) HEK293F cells were cultured in suspension to enter logarithmic phase; 3) performing cell electrotransfection, namely taking HEK293F cells in a logarithmic growth phase, uniformly mixing the HEK293F cells with transfection plasmids for electric shock, and culturing after the electric shock is finished to obtain the HEK293F cells;

the transfection plasmid can be an expression vector containing exogenous genes, and the exogenous genes can be any exogenous genes of which the expression products are nontoxic and harmless to cells, and include but not limited to exogenous genes such as expression antigens, antibodies, enzymes and the like; the expression vector can be any expression vector suitable for mammals, including but not limited to pcDNA, pIRES, pINFUSE-hIgG-Fc and the like;

in the electric shock step, the electric shock condition is that under the voltage of 200-300V, the electric shock is carried out for 1-2 times, and the electric shock duration is 10ms-20 ms; wherein the voltage may preferably be 230V, 250V, 280V, 300V, further, the voltage may more preferably be 230V; the number of shocks is preferably 2, wherein each shock is separated by 0.1-10s, wherein the separation time may preferably be 1s, 3s, 5s, and further, the separation time may more preferably be 1 s; the shock duration may preferably be 10ms, 15ms, 20ms, 25ms, and further, the shock duration may more preferably be 20 ms;

further, the electric shock condition is that the electric shock is carried out for 20ms under the voltage of 230V, and the electric shock is continuously carried out twice, wherein the interval of each time is 1 s;

HEK293F can greatly improve the survival rate and the transduction rate of cells under the condition of electric shock, and can obtain higher transduction rate of positive cells under the more optimal condition;

further, before electrotransfection, the method also comprises the steps of centrifuging the HEK293F cells to remove old culture medium, adjusting the concentration of the HEK293F cells by using fresh basal culture medium, and then adding transfection plasmid for uniformly mixing;

further, the HEK293F cells were mixed with the transfection plasmid prior to electrotransfection without the need for an incubation step at 4 ℃;

further, in the electrotransfection method, an electrotransfection buffer solution is not required to be used;

furthermore, in the electrotransfection method, the step of replacing a culture medium used by the cells after electrotransfection is not needed, and only L-glutamine or L-alanyl-L-glutamine required by cell growth is needed to be added, so that the electrically shocked HEK293F cells can be cultured.

In the invention, the mixed solution of the cells and the DNA before electrotransformation does not need to be incubated at 4 ℃; the method has the advantages that only a normal culture medium is used during electrotransfer, electrotransfection buffer solution is not required to be prepared and used, high transfection efficiency can still be obtained after electrotransfer, and the problems that in the prior art, the electrotransfer buffer solution is required to be prepared during electrotransfer, incubation is carried out at 4 ℃ before electrotransfer and the like are complex, and the consumption of nucleic acid during electrotransfer is large are solved. Furthermore, the electrotransfection method related by the application does not need to be replaced by a culture medium used for cells after electrotransfection, so that the operation is simpler and more convenient, the time is further saved, and the cost is saved. The electrotransfection method can be used for transfecting vectors expressing different target proteins into HEK293F suspension cells to realize the mass expression of the required proteins in a eukaryotic system. A sequence for coding a secretion signal peptide is added into an expression vector, so that the expressed protein can be secreted to the outside of the cell, and the subsequent purification work of a large amount of expressed protein and target protein of the cell is facilitated.

Drawings

FIG. 1 shows the cell viability under different voltage treatments.

FIG. 2 shows the transfection efficiency for different pulse numbers.

FIG. 3 shows the transfection efficiency at different intervals for two pulses.

FIG. 4 shows different pulse lengths versus electrotransfection efficiency.

FIG. 5 is the result of transfection under optimal electrotransfection conditions.

Detailed Description

First, HEK293F electrotransfection method

1.1 construction of pTT5-EGFP expression vector

Cloning EGFP gene to eukaryotic expression vector pTT5, introducing EcoR I and Not I enzyme cutting sites at the 5 'end and the 3' end of EGFP respectively through primer design, introducing a kozak sequence and an initiation codon at the 5 'end, and introducing a stop codon at the 3' end.

The primers were designed as follows:

F：5’CCGGAATTCGCCACCATGCACCACCACCACCACCACATGGTGAGCAAGGGCGAGG3’

R：5’TTGCGGCCGCTTACTTGTACAGCTCGTCCATG3’

PCR was performed using pCDNA3.1-EGFP as a template and the above primers. And performing gel recovery after electrophoresis of the PCR product to obtain the EGFP fragment. The vector pTT5 and the target segment EGFP are cut by two restriction enzymes EcoR I and Not I for 30min at 37 ℃, and after electrophoresis, gel recovery is carried out to obtain a double-enzyme digestion product. The fragments were incubated with the linearized pTT5 vector using T4 DNA ligase for 15min at 22 ℃ and subsequently transformed into competent cells DH 5. alpha. by heat treatment at 42 ℃ for 45s, recovered by incubation at 37 ℃ for 1h and plated onto LB plates (containing ampicillin) for selection, and incubated overnight at 37 ℃. And selecting a single colony for colony PCR and double enzyme digestion identification positive cloning the next day, culturing the screened positive strain, extracting plasmid, and finally performing gene sequencing identification.

And (3) culturing the strains which are successfully sequenced at 37 ℃ overnight, extracting the recombinant pTT5-EGFP plasmid by using an endotoxin-free kit the next day, finally dissolving the plasmid in sterile endotoxin-free water, and measuring the nucleic acid concentration by using Nanodrop.

1.2HEK293F cell suspension culture

HEK293F cells to be resuscitated were quickly removed from the liquid nitrogen tank and placed in a 37 ℃ water bath to thaw within 2 minutes of slow shaking. The surface of the frozen tube is sterilized by alcohol and then placed in a super clean bench, the cells are transferred into a 15ml centrifuge tube containing 8ml of HEK293F basic culture medium by a pipette gun, and the centrifuge is carried out for 5 minutes at 1000 rpm.

The HEK293F basal medium in this example contained 2mM L-alanyl-L-glutamine, e.g.using the commercially available GlutaMax^TM-I products. Alternatively, the HEK293F basal medium may also contain L-glutamine. L-Glutamine (L-Glutamine) is one of essential amino acids for cell growth, and can be used as an energy source for culturing cellsInvolved in protein synthesis and nucleic acid metabolism. However, L-glutamine is unstable in solution, for example, has a half-life of 3 weeks at 4 ℃ in a culture solution and 1 week at 37 ℃, and its degradation produces ammonia toxic to cells, and thus it is necessary to add it to a culture medium at the time of use. L-alanyl-L-glutamine is a dipeptide substitute for L-glutamine, and the decomposition products are L-alanyl and L-glutamine, which have no toxicity to cells. E.g. using GlutaMax^TMthe-I can be used as a direct substitute of L-glutamine and can be used in adherent and suspension mammalian cell culture. L-alanyl-L-glutamine (e.g. GlutaMax)^TM-I) has high solubility, thermostability (24 months at room temperature), improves growth efficiency and performance of mammalian cell culture systems, and also eliminates problems associated with the spontaneous decomposition of L-glutamine to ammonia during incubation, allowing for pre-addition and longer lasting culture. In this example, the HEK293F basal medium is preferably made to contain L-alanyl-L-glutamine.

After the cells were transferred into 15ml centrifuge tubes containing 8ml of HEK293F basal medium using the pipette gun as described above and centrifuged at 1000rpm for 5 minutes, the following procedure was continued: the supernatant was discarded, the cells were dispersed by flicking the bottom of the centrifuge tube with a finger, and after adding 2ml of the medium, the cells were gently blown with a pipette to disperse the cells. The cells were transferred to prepared 125ml shake flasks and 18ml HEK293F basal medium was added. The 125ml shake flask was placed in a 5% carbon dioxide incubator and incubated at 37 ℃ for 24h with a shaker at 130 rpm. Counting under an automatic Luna cell counter until the cell density reaches 2-3 × 10⁶cells/ml, indicating that the cells enter logarithmic growth phase, should be passaged.

After gently pipetting the cells, the cell suspension was transferred into a sterile centrifuge tube. Centrifuge at 1000rpm for 5 min. The supernatant was discarded and medium was added to bring the cell density to 0.5X 10⁶cells/ml. Transferring the diluted cell suspension into a shake flask, transferring into a carbon dioxide culture shaking table for shake culture (the conditions are the same), and performing cell electrotransfer after passage for three times.

1.3 cell electrotransfection

HEK293F cells were removed in logarithmic growth phase and centrifugedOld medium, fresh L-alanyl-L-glutamine-free (GlutaMax), respectively^TMHEK293F basal Medium of-I) to adjust the cell concentration to 5X 10⁶cells/ml. 250 mu L of cell and DNA mixed solution is put into a 0.4mm electric shock cup and is respectively shocked by DC for 15ms at 200V, 230V, 250V, 280V and 300V, and the electric shock is continuously carried out twice with the interval of 10s each time. After the electric shock is finished, standing for 10min, and measuring the cell viability, wherein the result is shown in figure 1. The optimum electrotransfection voltage is the cell survival rate of about 50%. As can be seen from FIG. 1, the 230V voltage is closest to about 50% survival rate, followed by 300V, 280V and 250V voltages.

HEK293F cells were removed in logarithmic growth phase, the old medium was removed by centrifugation, and fresh L-alanyl-L-glutamine-free (GlutaMax) was used separately^TMHEK293F basal Medium of-I) to adjust the cell concentration to 5X 10⁶cells/ml, 20. mu.g/ml pTT5-EGFP plasmid was added and mixed well. 500 microliter of cell and DNA mixed solution is put into a 0.4mm electric shock cup, one tube is shocked by 230V DC for 15ms, and the shock is carried out once; the other tube was shocked at 230V DC for 15ms, twice (two pulses) in succession, each time with an interval of 10 s. After the electric shock is finished, the cells are placed in a 5% carbon dioxide incubator at 130rpm and cultured at 37 ℃, the total number of the cells and the number of the cells with fluorescence are measured after 48 hours, and the positive cell rate is calculated, and the result is shown in figure 2. At 15ms of 230V DC shock, two shocks (two pulses) are continuously applied, each time with the interval of 10s, and the transfection effect is better.

HEK293F cells were removed in logarithmic growth phase, the old medium was removed by centrifugation, and fresh L-alanyl-L-glutamine-free (GlutaMax) was used separately^TMHEK293F basal Medium of-I) to adjust the cell concentration to 5X 10⁶cells/ml, 20. mu.g/ml pTT5-EGFP plasmid was added and mixed well. 500 mul of the mixed solution of the cells and the DNA is placed in a 0.4mm electric shock cup, and the electric shock is continuously carried out twice at the DC voltage of 230V for 15ms, wherein the interval of each electric shock is 0.1s, 0.5s, 1s, 3s, 5s and 10 s. After the electric shock is finished, the cells are placed in a 5% carbon dioxide incubator at 130rpm and cultured at 37 ℃, the total number of the cells and the number of the cells with fluorescence are measured after 48 hours, and the positive cell rate is calculated, and the result is shown in figure 3. At 15ms of 230V DC shock, two shocks (two pulses) in succession, each at 1s interval, had optimal transfection effect, and secondly at 3s and 5s intervals of the same typeHas better transfection effect.

HEK293F cells were removed in logarithmic growth phase, the old medium was removed by centrifugation, and fresh L-alanyl-L-glutamine-free (GlutaMax) was used separately^TMHEK293F basal Medium of-I) to adjust the cell concentration to 5X 10⁶cells/ml, 20. mu.g/ml pTT5-EGFP plasmid was added and mixed well. 500 μ L of the mixed solution of the cells and the DNA is placed in a 0.4mm electric shock cup, and electric shock is continuously carried out twice at intervals of 1s for 10ms, 15ms, 20ms and 25ms at 230V DC. After the electric shock is finished, the cells are placed in a 5% carbon dioxide incubator at 130rpm and cultured at 37 ℃, after 48 hours, the total number of the cells and the number of the cells with fluorescence are measured, and the positive cell rate is calculated, and the result is shown in figure 4. At 230V DC shock, two shocks in succession (two pulses) each spaced 1s apart, with optimal transfection efficiency at 20ms duration, followed by equally good transfection efficiency at intervals 10s and 15 s.

According to the results of the above electrotransformation experiments, L-alanyl-L-glutamine (GlutaMax) was not added^TMHEK293F basal Medium of-I) to adjust the cell concentration to 5X 10⁶cells/ml, 20. mu.g/ml pTT5-EGFP plasmid was added and mixed well. 500 mul of the mixed solution of the cells and the DNA are placed in a 0.4mm electric shock cup, the DC electric shock is carried out for 20ms at 230V, the electric shock is continuously carried out twice, under the condition that the interval of each time is 1s, the positive cell rate is 84 percent, and the result is shown in figure 5.

While only certain embodiments of the present invention have been illustrated and described, it will be apparent to those skilled in the art that various changes and modifications can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. An electric transfection method for HEK293F suspension cells is characterized in that the electric transfection method is adopted to shock the HEK293F under the conditions that the electric shock is 1-2 times under the voltage of 200-300V and the duration time of the electric shock is 10-20 ms.

2. An electrotransfection method of HEK293F suspension cells, which comprises the following steps:

1) preparing a transfection plasmid; 2) HEK293F cells were cultured in suspension to enter logarithmic phase; 3) taking HEK293F cells in logarithmic phase, mixing the HEK293F cells with transfection plasmids uniformly for electric shock, and culturing after the electric shock is finished;

the electric shock condition is that under the voltage of 200-300V, 1-2 electric shocks are carried out, and the electric shock duration is 10-20 ms.

3. The method of any of claims 1-2 for electrotransfection of HEK293F suspension cells at a voltage selected from 230V, 250V, 280V or 300V.

4. The method of any of claims 1-3 for electrotransfection of HEK293F suspension cells to 2 shocks.

5. The method of claim 4 wherein the time interval between two shocks is 0.1-10 seconds.

6. The method of any of claims 4 and 5 for electrotransfection of HEK293F suspension cells with a time interval of 1s, 3s or 5s between two shocks.

7. The method of any of claims 1-6 wherein the shock duration is 10ms, 15ms, 20ms or 25 ms.

8. The method for electrotransfection of HEK293F suspension cells of any one of claims 1 to 7, wherein the shock to HEK293F is administered after mixing HEK293F with the transfection plasmid.

9. The method of claim 8 wherein the transfection plasmid is an expression vector comprising a foreign gene.

10. The method for electrotransfection of HEK293F suspension cells of claim 9 wherein the foreign gene is any foreign gene whose expression product is not toxic or harmful to the cells themselves.

11. The method of claim 10 wherein the foreign gene comprises a foreign gene that expresses one or more of an antigen, an antibody, or an enzyme.

12. The method of any of claims 9-11 wherein the expression vector is selected from any expression vector suitable for mammalian use.

13. The method for electrotransfection of HEK293F suspension cells of claim 12 wherein the expression vector is selected from any expression vector of pcDNA, pIRES or pINFUSE-hIgG-Fc.

14. The method for electrotransfection of HEK293F suspension cells according to any one of claims 1 to 13, further comprising the step of centrifuging HEK293F cells to remove old medium, adjusting HEK293F cell concentration in fresh basal medium, adding transfection plasmid and mixing.

15. The method of any of claims 1-14 wherein no incubation step at 4 ℃ is required prior to electrotransfection.

16. The method of any of claims 1-15 for electrotransfection of HEK293F suspension cells, wherein the electrotransfection method does not require the use of an electrotransfection buffer.

17. The method for electrotransfection of HEK293F suspension cells according to any one of claims 1 to 16, wherein after electrotransfection, the electrically shocked HEK293F cells can be cultured by adding L-glutamine or L-alanyl-L-glutamine required for cell growth without the need to replace the culture medium used for the cells.