CN114438131A - 293 cell transfection method - Google Patents
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Abstract
The invention discloses a transfection method of 293 cells, which comprises the following steps: mixing 293 cells with a substance to be transfected, and carrying out flow electrotransfection, wherein the voltage value of the flow electrotransfection is 100-300V, the pulse value is 1000-5000 mus, the electric shock frequency is 1-10 times, the interval is 100-600 ms, the flow rate of a transfection solution is 1-10 mL/min, and the cell density of the 293 cells during transfection is 1 multiplied by 105cells/mL～5×107cells/mL; the transfected 293 cells are inoculated to a transfection medium for culture, the transfection medium contains L-tyrosine, nicotinamide, magnesium sulfate and hydrocortisone, and the flow electrotransfection condition and the nutrition environment after transfection are matched with each other, so that the transfection damage of the cells can be repaired, a better growth environment can be obtained, and higher gene expression strength, cell density and cell survival rate can be realized.
Description
Technical Field
The invention relates to the technical field of transfection media, in particular to a transfection method of 293 cells.
Background
In the development of biological medicines, the target gene plasmid is often transfected into a host cell for culture so as to obtain the target biological medicine. The flow electrotransformation technology is a high-efficiency transfection technology, can generate a uniform electric field with enough strength under lower voltage, and can realize high transfection efficiency. The transfection medium can better protect cells, reduce the cell death rate and obviously improve the cell transfection rate. The host cells used in large quantities at first are prokaryotic expression systems, and the eukaryotic expression systems are limited in application due to technical problems and are inferior to the prokaryotic expression systems. In recent years, due to the progress of technology and the unique advantages of eukaryotic expression systems, the original prokaryotic system is replaced in some fields. For example, some research institutions use eukaryotic 293 cells for the production of protein drugs or other biopharmaceuticals.
293 cells (human embryonic kidney cells, also called HEK cells) are easy to transfect and are cell lines which are commonly used for expression research of foreign genes. Moreover, because of the translation and modification functions of 293 cells to proteins, the potency of the activity of the drug is better. Because of the structural difference between eukaryotic cells and prokaryotic cells, the culture conditions are not the same in adaptability, the required transfection conditions are different, and the prokaryotic cell transfection conditions are not suitable for eukaryotic cells. Because the cell transfection process can cause the change of the cell membrane surface and cause the stimulation to the cells, the cells are fragile after transfection, and the requirements of cell repair and transfection substance expression after transfection on the culture environment are higher. In addition to the growth health of the cells, different transfection methods, conditions and culture methods after transfection play an important role for the effect of transfection. The research on eukaryotic cell transfection in the market is very limited, and especially the development of flow electrotransformation schemes for eukaryotic cells is much less and less.
In view of this, it is necessary to develop a flow electroporation protocol suitable for 293 cells.
Disclosure of Invention
Based on the above, the invention provides a flow electrotransfer method capable of realizing high-efficiency transfection to transfect 293 cells.
The invention aims to provide a transfection method of 293 cells, which comprises the following steps:
mixing 293 cells with a substance to be transfected, and carrying out flow electrotransfection, wherein the voltage value of the flow electrotransfection is 100-300V, the pulse value is 1000-5000 mus, the electric shock frequency is 1-10 times, the interval is 100-600 ms, the flow rate of a transfection solution is 1-10 mL/min, and the cell density of the 293 cells during transfection is 1 multiplied by 105cells/mL～5×107cells/mL；
And inoculating the transfected 293 cells into a transfection medium to culture, wherein the transfection medium contains L-tyrosine, nicotinamide, magnesium sulfate and hydrocortisone.
In one embodiment, the voltage value of the flow electrotransfection is 160V-250V, the pulse value is 1000 mus-2000 mus, the electric shock frequency is 3-6 times, the interval is 200 ms-400 ms, and the flow rate of the transfection liquid is 3.5 mL/min-5.5 mL/min.
In one embodiment, the cell density of the 293 cells at transfection is 1X 106cells/mL～5×107cells/mL。
In one embodiment, the transfection medium is a serum-free transfection medium comprising the following components at the following concentrations:
0.1843 g/L-tyrosine-0.396 g/L,
0.1704 g/L-0.1874 g/L of asparagine,
0.3361 g/L-cystine-0.3697 g/L,
0.1722 g/L-0.1895 g/L-glutamic acid,
0.1052 g/L-0.1157 g/L isoleucine,
0.1322 g/L-0.1454 g/L leucine,
0.0903 g/L-0.0993 g/L methionine,
0.0940 g/L-0.1034 g/L phenylalanine,
0.0521 g/L-0.0573 g/L proline,
0.0940 g/L-0.1034 g/L serine,
Threonine 0.1518 g/L-0.1669 g/L,
0.4255-0.4680 g/L of tryptophan,
Valine of 0.1508g/L to 0.1659g/L,
0.0010 g/L-0.0011 g/L of choline chloride,
5.5860 g/L-6.1446 g/L of glucose,
0.0014 g/L-0.0016 g/L of nicotinamide,
0.00015 g/L-0.00025 g/L of riboflavin,
Vitamin B120.0009g/L-0.0010 g/L,
Pyridoxal 0.0112-0.0123 g/L,
Thiamine hydrochloride 0.0033-0.0036 g/L,
0.00025 g/L-0.00035 g/L of biotin,
Inositol 0.0009-0.0010 g/L,
0.2346 g/L-0.2581 g/L potassium chloride,
0.1080-0.1188 g/L magnesium chloride,
0.0200 g/L-0.0369 g/L magnesium sulfate,
0.0205 g/L-0.0225 g/L of sodium dihydrogen phosphate,
0.0326 g/L-0.0358 g/L of disodium hydrogen phosphate,
1.8732-2.0605 g/L of sodium bicarbonate,
0.00055g/L to 0.00065g/L of zinc sulfate heptahydrate,
Linoleic acid 0.00015-0.00025 g/L,
0.00025 g/L-0.00035 g/L of sodium selenite,
0.0001-0.0002 g/L of hydrocortisone,
0.0003 g/L-0.0004 g/L of lipoic acid,
0.0633 g/L-0.0696 g/L of sodium pyruvate.
The invention provides a 293 cell transfection method, which adopts flow electrotransfection, and sets proper electrotransfection conditions, wherein the voltage value is 100V-300V, the pulse value is 1000 mus-5000 mus, the electric shock frequency is 1-10 times, the interval is 100 ms-600 ms, the flow rate of a transfection solution is 1 mL/min-10 mL/min, and the cell density of the 293 cell during transfection is 1 multiplied by 105cells/mL～5×107cells/mL, and culturing in a proper transfection medium after flow electrotransformation, wherein the flow electrotransformation condition and the transfection medium condition are matched with each other, so that stimulation of flow electrotransformation can be relieved, and higher gene expression intensity, cell density and cell survival rate can be realized after transfection.
Drawings
FIG. 1 is a graph of the fluorescence intensity of proteins expressed by HEK293 cells cultured in different serum-free transfection media according to example 1 of the present invention;
FIG. 2A shows the cell density of HEK293 cells cultured in different serum-free transfection media according to example 1 of the present invention;
FIG. 2B is a graph of cell viability of HEK293 cells cultured with different serum-free transfection media according to example 1 of the present invention;
FIG. 3 is a graph showing the fluorescence intensity of HEK293 cell expressed protein cultured in different serum-free transfection media according to examples 2-5 of the present invention;
FIG. 4A is a cell density map of HEK293 cells cultured with different serum-free transfection media according to examples 2-5 of the present invention;
FIG. 4B is a graph showing the viability of HEK293 cells cultured in different serum-free transfection media according to examples 2-5 of the present invention;
FIG. 5 is a graph comparing the fluorescence intensity of HEK293 cells cultured before and after optimization of the serum-free transfection medium in accordance with the embodiments of the present invention.
FIG. 6 is a graph showing the comparison of fluorescence intensity of proteins expressed by HEK293 cells in example 6 of the present invention.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In the present invention, "first aspect", "second aspect", "third aspect" and the like are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor are they to be construed as implicitly indicating the importance or quantity of the technical feature indicated.
As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. As used herein, the terms "comprising," "including," and "comprising" are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps. In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
In the present invention, the numerical range is defined to include both end points of the numerical range unless otherwise specified.
The percentage contents referred to in the present invention mean, unless otherwise specified, mass percentages for solid-liquid mixing and solid-solid phase mixing, and volume percentages for liquid-liquid phase mixing.
The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system to which the component is added.
The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a certain temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.
The embodiment of the invention provides a transfection method of 293 cells, which comprises the following steps:
mixing 293 cells with a substance to be transfected, and carrying out flow electrotransfection, wherein the voltage value of the flow electrotransfection is 100-300V, the pulse value is 1000-5000 mus, the electric shock frequency is 1-10 times, the interval is 100-600 ms, the flow rate of a transfection solution is 1-10 mL/min, and the cell density of the 293 cells during transfection is 1 multiplied by 105cells/mL～5×107cells/mL；
And inoculating the transfected 293 cells into a transfection medium to culture, wherein the transfection medium contains L-tyrosine, nicotinamide, magnesium sulfate and hydrocortisone.
The invention provides a 293 cell transfection method, which adopts flow electrotransfection, and sets proper electrotransfection conditions, wherein the voltage value is 100V-300V, the pulse value is 1000 mus-5000 mus, the electric shock frequency is 1-10 times, the interval is 100 ms-600 ms, the flow rate of a transfection solution is 1 mL/min-10 mL/min, and the cell density of the 293 cell during transfection is 1 multiplied by 105cells/mL～5×107cells/mL, and in-flow electricAfter the transfection, the cells are cultured in a proper transfection medium, the flow electrotransformation condition and the transfection medium condition are matched with each other, so that the stimulation of the flow electrotransformation can be relieved, and higher gene expression intensity, cell density and cell survival rate can be realized after transfection.
As described herein, "substance to be transfected" refers to a substance transfected into 293 cells, such as nucleic acids (DNA/RNA)/carbohydrates or proteins, etc.
As described herein, "seeding" refers to a process of supplying a cell culture to a bioreactor or another container. The cells may be previously propagated in another bioreactor or vessel. Or the cells are frozen and thawed prior to being supplied to the bioreactor or vessel. The term is used to refer to any number of cells, including a single cell.
As described herein, "medium" refers to a solution containing nutrients that nourish the growth of cells. These solutions provide essential and non-essential nutrients required for substantial cell growth and/or survival. The solution may also contain components that promote growth and/or survival above the minimum rate. The medium may be a "chemically defined medium" -a serum-free medium free of proteins, hydrolysates, or unknown composition components. The chemically defined medium does not contain animal-derived components, and all of its components have known chemical structures.
As described herein, "cell density" refers to the number of cells present in a specified volume of culture medium.
As described herein, "cell viability" refers to the ability of cells in culture to survive under a set of specified culture conditions or test variables. The term as used herein also refers to the relationship between the fraction of cells that survive a particular time and the total number of live and dead cells in the culture at that time.
The 293 cells described herein are Human Embryonic Kidney (HEK) cells.
293 cells are human kidney epithelial cell lines, have a plurality of derivative strains, such as HEK293, 293T/17 and the like, are derived from human embryonic kidney cells, rarely express endogenous receptors required by extracellular ligands, are relatively easy to transfect, and are cell strains which are commonly used for expressing and researching exogenous genes.
Wherein the HEK cells are selected from: any one of HEK293, HEK 293T, HEK 293S, and HEK293 EBNA.
The working principle of the flow type electrotransfer instrument is that the mixed solution of cells and special buffer solution for electrotransfer continuously flows through a pulse electric field, and the voltage, the pulse width (namely the duration time, microsecond precision) and the pulse interval (millisecond precision) of pulse current are precisely controlled to enable protein molecules on cell membranes to displace to form a plurality of micropores, so that exogenous molecules/substances (such as DNA, RNA, protein and the like) can enter the interior of the cells through the micropores of the cell membranes under the action of the electric field.
The flow electrotransformation technology can generate a uniform electric field with enough strength under lower voltage, and can realize high transfection rate and high cell survival rate; the transfection expression culture medium can better protect cells, reduce the cell death rate and obviously improve the cell transfection rate; the protein expression culture medium can greatly improve the protein expression from the analysis of cell metabolism.
In some embodiments, the voltage value of the flow electrotransfection is 160V-250V, the pulse value is 1000-2000 mus, the number of electric shocks is 3-6, the interval is 200-400 ms, and the flow rate of the transfection solution is 3.5-5.5 mL/min.
In some embodiments, the 293 cell has a cell density of 1X 10 when added with a substance to be transfected6cells/mL～5×107cells/mL. For example, it may be 1 × 106cells/mL、2×106cells/mL、3×106cells/mL、4×106cells/mL、5×106cells/mL、6×106cells/mL、7×106cells/mL、8×106cells/mL、9×106cells/mL、1×107cells/mL、1.5×107cells/mL、2×107cells/mL、2.5×107cells/mL、3×107cells/mL、3.5×107cells/mL、4×107cells/mL、4.5×107cells/mL、5×107cells/mL。
In some embodiments, the pulse value is 1 at a voltage value of 210V000 μ s, 6 times of electric shock, 4.5mL/min of flow rate, 350ms of interval, 48h of cell growth before transfection, and 4X 10 of cell density during transfection7cells/mL obtained by transfection.
In some embodiments, the transfection medium is a serum-free transfection medium comprising the following concentrations of components:
0.1843 g/L-tyrosine-0.396 g/L,
0.1704 g/L-0.1874 g/L of asparagine,
0.3361 g/L-cystine-0.3697 g/L,
0.1722 g/L-0.1895 g/L-glutamic acid,
0.1052 g/L-0.1157 g/L isoleucine,
0.1322 g/L-0.1454 g/L leucine,
0.0903 g/L-0.0993 g/L methionine,
0.0940 g/L-0.1034 g/L phenylalanine,
0.0521 g/L-0.0573 g/L proline,
0.0940 g/L-0.1034 g/L serine,
Threonine 0.1518 g/L-0.1669 g/L,
0.4255-0.4680 g/L of tryptophan,
Valine of 0.1508g/L to 0.1659g/L,
0.0010 g/L-0.0011 g/L of choline chloride,
5.5860 g/L-6.1446 g/L of glucose,
0.0014 g/L-0.0016 g/L of nicotinamide,
0.00015 g/L-0.00025 g/L of riboflavin,
Vitamin B120.0009g/L-0.0010 g/L,
Pyridoxal 0.0112-0.0123 g/L,
Thiamine hydrochloride 0.0033-0.0036 g/L,
0.00025 g/L-0.00035 g/L of biotin,
Inositol 0.0009-0.0010 g/L,
0.2346 g/L-0.2581 g/L potassium chloride,
0.1080-0.1188 g/L magnesium chloride,
0.0200 g/L-0.0369 g/L magnesium sulfate,
0.0205 g/L-0.0225 g/L of sodium dihydrogen phosphate,
0.0326 g/L-0.0358 g/L of disodium hydrogen phosphate,
1.8732 g/L-2.0605 g/L of sodium bicarbonate,
0.00055g/L to 0.00065g/L of zinc sulfate heptahydrate,
Linoleic acid 0.00015-0.00025 g/L,
0.00025 g/L-0.00035 g/L of sodium selenite,
0.0001-0.0002 g/L of hydrocortisone,
0.0003 g/L-0.0004 g/L of lipoic acid,
0.0633 g/L-0.0696 g/L of sodium pyruvate.
The transfection medium comprises amino acid, salt, trace elements and other additives. Through the overall design of the type and the content of the formula, the 293 cell transfected by the flow electrotransformation method is suitable for culture after transfection. The 293 cells are transfected and then inoculated to the culture medium for culture, so that the stimulation of flow electrotransformation can be relieved, and higher gene expression strength, cell density and cell survival rate can be realized after transfection.
In some embodiments, the transfection medium comprises the following concentrations of components:
0.198 g/L-0.396 g/L of L-tyrosine,
0.1704 g/L-0.1830 g/L of asparagine,
0.3361 g/L-cystine-0.3610 g/L,
0.1722 g/L-0.1850 g/L-glutamic acid,
0.1052 g/L-0.1130 g/L isoleucine,
0.1322 g/L-0.1420 g/L leucine,
0.0903 g/L-0.0970 g/L methionine,
0.0940 g/L-0.1010 g/L phenylalanine,
0.0521 g/L-0.0560 g/L proline,
0.0940 g/L-0.1010 g/L serine,
Threonine 0.1518 g/L-0.1630 g/L,
0.4255-0.4570 g/L of tryptophan,
Valine of 0.1508g/L to 0.1620g/L,
0.0010 g/L-0.0011 g/L of choline chloride,
5.5860 g/L-6.0000 g/L of glucose,
0.0014 g/L-0.0016 g/L of nicotinamide,
0.00018 g/L-0.00022 g/L of riboflavin,
Vitamin B120.0009g/L-0.0010 g/L,
0.0112 g/L-0.01230 g/L of pyridoxal,
Thiamine hydrochloride 0.0033-0.0035 g/L,
0.00028 g/L-0.00032 g/L of biotin,
Inositol 0.0009-0.0010 g/L,
0.2346 g/L-0.2520 g/L potassium chloride,
0.1080-0.1160 g/L magnesium chloride,
0.0200 g/L-0.0369 g/L magnesium sulfate,
0.0205 g/L-0.0220 g/L of sodium dihydrogen phosphate,
0.0326 g/L-0.0350 g/L of disodium hydrogen phosphate,
1.8732-2.0120 g/L of sodium bicarbonate,
0.00058g/L to 0.00062g/L of zinc sulfate heptahydrate,
Linoleic acid 0.00018 g/L-0.00022 g/L,
0.00028 g/L-0.00032 g/L of sodium selenite,
0.0001-0.0015 g/L hydrocortisone,
0.0003 g/L-0.0004 g/L of lipoic acid,
0.0633 g/L-0.0680 g/L of sodium pyruvate.
In some preferred embodiments:
the asparagine is 0.1820 g/L-0.1830 g/L;
the L-cystine is 0.3600 g/L-0.3610 g/L;
the L-glutamic acid is 0.1840 g/L-0.1850 g/L;
the isoleucine is 0.1120 g/L-0.1130 g/L;
the leucine is 0.1410 g/L-0.1420 g/L;
the methionine is 0.0960 g/L-0.0970 g/L;
the phenylalanine is 0.1000 g/L-0.1010 g/L;
proline is 0.0550 g/L-0.0560 g/L;
serine is 0.1000 g/L-0.1010 g/L;
threonine is 0.1620 g/L-0.1630 g/L;
the tryptophan is 0.4560 g/L-0.4570 g/L;
the valine is 0.1610 g/L-0.1620 g/L;
choline chloride is 0.0010 g/L-0.0011 g/L;
the glucose is 5.9000 g/L-6.0000 g/L;
the riboflavin is 0.00021g/L to 0.00022 g/L;
vitamin B12 is 0.0009 g/L-0.0010 g/L;
the pyridoxal is 0.01220 g/L-0.01230 g/L;
thiamine hydrochloride is 0.0034g/L to 0.0035 g/L;
biotin is 0.00028 g/L-0.00032 g/L;
inositol is 0.0009 g/L-0.0010 g/L;
the potassium chloride is 0.2510 g/L-0.2520 g/L;
the magnesium chloride is 0.1150 g/L-0.1160 g/L;
the sodium dihydrogen phosphate is 0.0219 g/L-0.0220 g/L;
the content of the disodium hydrogen phosphate is 0.0340 g/L-0.0350 g/L;
the sodium bicarbonate is 2.0110 g/L-2.0120 g/L;
the zinc sulfate heptahydrate is 0.00061 g/L-0.00062 g/L;
linoleic acid is 0.00021 g/L-0.00022 g/L;
the sodium selenite accounts for 0.00031 g/L-0.00032 g/L;
the lipoic acid is 0.00039 g/L-0.0004 g/L;
the sodium pyruvate is 0.0670 g/L-0.0680 g/L.
Preferably, the concentration of L-tyrosine in the transfection medium is 0.198 g/L-0.208 g/L. More preferably, it is 0.198 to 0.199 g/L.
Preferably, the concentration of nicotinamide in the transfection medium is 0.0015g/L to 0.0016 g/L.
Preferably, the concentration of the magnesium sulfate in the transfection medium is 0.026 g/L-0.036 g/L. More preferably, it is 0.035 to 0.036 g/L.
Preferably, the concentration of the hydrocortisone in the transfection medium is 0.0009 g/L-0.0010 g/L.
In some embodiments, the transfection medium further comprises glutamine at a concentration of 0.4g/L to 0.5 g/L.
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. It will be appreciated by those skilled in the art that various other changes, modifications, substitutions, combinations, and omissions may be made in the form and detail of the invention without departing from the spirit and scope of the invention.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
EXAMPLE 1 selection of transfection Medium
Taking out the suspension HEK293 cells cultured in growth medium MEM for 48h, counting, transferring the cells into a centrifuge tube, centrifuging at 1000rpm for 5min, discarding the supernatant, washing the cells twice with DPBS to remove the growth medium, taking 5mL of transfection buffer solution to resuspend the cells, adjusting the cell density to 4 × 106cells/mL, adding plasmid, mixing, and transfecting. The voltage value is 200V, the pulse value is 600 mus, the electric shock times are 3 times, the flow rate is 4mL/min, the interval is 300ms, and the cell growth is 48h before transfection.
After transfection, the cells were incubated in an incubator at 37 ℃ for 20min, centrifuged at 1000rpm for 10min, the supernatant was discarded, and the cells were resuspended in serum-free transfection media of 4 different formulations in Table 1, respectively, such that the cells were 4X 106cells/mL were inoculated into cell culture flasks at 37 ℃ with 8% CO2，130And (3) culturing in a shaker incubator at rpm, and detecting the fluorescence intensity, cell density and cell viability of the expressed protein to measure the transfection efficiency.
The specific components of serum-free transfection media M1, M2, M3 and M4 used are shown in Table 1.
TABLE 1 serum-free transfection Medium formulations M1, M2, M3 and M4
The fluorescence intensity, cell density and viability were measured 48h after transfection in medium, and the results are shown in fig. 1, fig. 2A and fig. 2B, which indicate that HEK293 cells grow better in M2 and M3 medium and better in M2 medium after transfection.
EXAMPLE 2 optimization of L-tyrosine
The transfection method and conditions were substantially the same as those of example 1 except that the composition of the transfection medium was different.
The transfection medium of this example was optimized for L-tyrosine based M2 medium of example 1, and single factor experiments were performed according to the test concentrations in Table 2.
The fluorescence intensity, cell density and viability are detected after the serum-free transfection medium is cultured for 48 hours, and the results are shown in figure 3, figure 4A and figure 4B, which show that the L-tyrosine is important for culturing HEK293 cells after flow electrotransfection. The L-tyrosine concentration after transfection is 0.1980g/L, which is more suitable for the growth of HEK293 cells after transfection.
TABLE 2L-tyrosine optimization Experimental design
Example 3 optimization experiment of Nicotinamide
The transfection procedure and conditions were essentially the same as in example 1, except that the composition of the serum-free transfection medium was different.
The transfection medium of this example was a nicotinamide optimization experiment based on the CD3 medium of example 2, and a one-way experiment was performed according to the test concentrations in Table 3.
The fluorescence intensity, cell density and viability are detected after the culture medium is cultured for 48 hours in a serum-free transfection medium, and the results are shown in fig. 3, 4A and 4B, which indicate that nicotinamide is important for the culture of HEK293 cells after flow electrotransfection. Nicotinamide concentration of 0.0016g/L is more suitable for growth of HEK293 cells after transfection.
Table 3 nicotinamide optimization experimental design
Example 4 optimization experiment of magnesium sulfate
The transfection procedure and conditions were essentially the same as in example 1, except that the composition of the serum-free transfection medium was different.
The transfection medium of this example was optimized for magnesium sulfate based on the CD7 medium of example 3, and a one-way experiment was performed according to the test concentrations in Table 4.
The fluorescence intensity, cell density and viability are detected after the serum-free transfection medium is cultured for 48 hours, and the results are shown in fig. 3, fig. 4A and fig. 4B, and the results show that magnesium sulfate is important for culturing the HEK293 cell after flow electrotransfection. The magnesium sulfate concentration of 0.036g/L is more suitable for the growth of HEK293 cells after transfection.
Table 4 magnesium sulfate optimization experimental design
Example 5 optimization experiment of hydrocortisone
The transfection procedure and conditions were essentially the same as in example 1, except that the composition of the serum-free transfection medium was different.
The transfection medium of this example was optimized for hydrocortisone on the basis of the CD11 medium of example 4, and a one-way experiment was performed according to the test concentrations of Table 5.
The fluorescence intensity, cell density and viability were measured 48h after serum-free transfection medium culture, and the results are shown in fig. 3, fig. 4A and fig. 4B, which indicate that hydrocortisone is important for the culture of HEK293 cells after flow electrotransfection. The hydrocortisone concentration of 0.0010g/L is more suitable for the growth of HEK293 cells after transfection.
TABLE 5 hydrocortisone optimization experimental design
Through the optimization experiment of the culture medium, a chemically defined transfection culture medium which can enable the HEK293 cell strain to express the target protein efficiently and transiently is developed. Comparison of the results of the chemically defined transfection medium before and after optimization of L-tyrosine, nicotinamide, magnesium sulfate and hydrocortisone is shown in FIG. 5.
Example 6 optimization of flow electrotransfection conditions was performed on the basis of the transfection medium obtained in example 5.
The voltage value of the flow electrotransfection is 100V-300V, the pulse value is 1000 mus-5000 mus, the flow rate of the transfection solution is 1 mL/min-10 mL/min, and the cell density of the 293 cell during transfection is 1 multiplied by 105cells/mL～5×107cells/mL, single factor experiments were performed. The number of shocks was 6 with an interval of 350 ms.
TABLE 6 optimization experiment design of voltage values for flow electrotransfection
TABLE 7 pulse value optimization experiment design for flow electrotransfection
TABLE 8 transfection solution flow rate optimization experiment design for flow electrotransfection
TABLE 9 cell Density optimization experiment design of 293 cells during flow electrotransfection transfection
The results are shown in FIG. 6, and show that the cell density at transfection was 4X 10 at a transfection voltage value of 210V, a pulse value of 1000. mu.s, a shock frequency of 6 times, a flow rate of 4.5mL/min, an interval of 350ms, a cell growth time of 48h before transfection and a cell density at transfection of 4X 107cells/mL transfection gave better transfection results.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the patent protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the patent of the invention is subject to the appended claims, and the description can be used for explaining the contents of the claims.
Claims (10)
1.293 cell transfection method, characterized in that it comprises the following steps:
mixing 293 cells with a substance to be transfected, and carrying out flow electrotransfection, wherein the voltage value of the flow electrotransfection is 100-300V, the pulse value is 1000-5000 mus, the electric shock frequency is 1-10 times, the interval is 100-600 ms, the flow rate of a transfection solution is 1-10 mL/min, and the cell density of the 293 cells during transfection is 1 multiplied by 105cells/mL～5×107cells/mL；
And inoculating the transfected 293 cells into a transfection medium to culture, wherein the transfection medium contains L-tyrosine, nicotinamide, magnesium sulfate and hydrocortisone.
2. The transfection method for 293 cells of claim 1, wherein the voltage value of the flow electrotransfection is 160V-250V, the pulse value is 1000-2000 μ s, the number of electric shocks is 3-6, the interval is 200 ms-400 ms, and the flow rate of the transfection solution is 3.5 mL/min-5.5 mL/min.
3. A method for transfecting a 293 cell according to claim 1, wherein the cell density of the 293 cell at the time of transfection is 1 x 106cells/mL～5×107cells/mL。
4. A method of transfecting a 293 cell according to claim 1, wherein the transfection medium is a serum-free transfection medium comprising the following components at the following concentrations:
0.1843 g/L-tyrosine-0.396 g/L,
0.1704 g/L-0.1874 g/L of asparagine,
0.3361 g/L-cystine-0.3697 g/L,
0.1722 g/L-0.1895 g/L-glutamic acid,
0.1052 g/L-0.1157 g/L isoleucine,
0.1322 g/L-0.1454 g/L leucine,
0.0903 g/L-0.0993 g/L methionine,
0.0940 g/L-0.1034 g/L phenylalanine,
0.0521 g/L-0.0573 g/L proline,
0.0940 g/L-0.1034 g/L of serine,
Threonine 0.1518 g/L-0.1669 g/L,
0.4255-0.4680 g/L of tryptophan,
Valine of 0.1508g/L to 0.1659g/L,
0.0010 g/L-0.0011 g/L of choline chloride,
5.5860 g/L-6.1446 g/L of glucose,
0.0014 g/L-0.0016 g/L of nicotinamide,
0.00015 g/L-0.00025 g/L of riboflavin,
Vitamin B120.0009g/L-0.0010 g/L,
Pyridoxal 0.0112-0.0123 g/L,
Thiamine hydrochloride 0.0033-0.0036 g/L,
0.00025 g/L-0.00035 g/L of biotin,
Inositol 0.0009-0.0010 g/L,
0.2346 g/L-0.2581 g/L potassium chloride,
0.1080-0.1188 g/L magnesium chloride,
0.0200 g/L-0.0369 g/L magnesium sulfate,
0.0205 g/L-0.0225 g/L of sodium dihydrogen phosphate,
0.0326 g/L-0.0358 g/L of disodium hydrogen phosphate,
1.8732-2.0605 g/L of sodium bicarbonate,
0.00055g/L to 0.00065g/L of zinc sulfate heptahydrate,
Linoleic acid 0.00015-0.00025 g/L,
0.00025 g/L-0.00035 g/L of sodium selenite,
0.0001-0.0002 g/L of hydrocortisone,
0.0003 g/L-0.0004 g/L of lipoic acid,
Sodium pyruvate is 0.0633 g/L-0.0696 g/L.
5. A method of transfecting a 293 cell according to claim 4, wherein the transfection medium comprises the following components at the following concentrations:
0.198 g/L-0.396 g/L of L-tyrosine,
0.1704 g/L-0.1830 g/L of asparagine,
0.3361 g/L-cystine-0.3610 g/L,
0.1722 g/L-0.1850 g/L-glutamic acid,
0.1052 g/L-0.1130 g/L isoleucine,
0.1322 g/L-0.1420 g/L leucine,
0.0903 g/L-0.0970 g/L methionine,
0.0940 g/L-0.1010 g/L phenylalanine,
0.0521 g/L-0.0560 g/L proline,
0.0940 g/L-0.1010 g/L serine,
Threonine 0.1518 g/L-0.1630 g/L,
0.4255-0.4570 g/L of tryptophan,
Valine of 0.1508g/L to 0.1620g/L,
0.0010 g/L-0.0011 g/L of choline chloride,
5.5860 g/L-6.0000 g/L of glucose,
0.0014 g/L-0.0016 g/L of nicotinamide,
0.00018 g/L-0.00022 g/L of riboflavin,
Vitamin B120.0009g/L-0.0010 g/L,
0.0112 g/L-0.01230 g/L of pyridoxal,
Thiamine hydrochloride 0.0033-0.0035 g/L,
0.00028 g/L-0.00032 g/L of biotin,
Inositol 0.0009-0.0010 g/L,
0.2346 g/L-0.2520 g/L potassium chloride,
0.1080-0.1160 g/L magnesium chloride,
0.0200 g/L-0.0369 g/L magnesium sulfate,
0.0205 g/L-0.0220 g/L of sodium dihydrogen phosphate,
0.0326 g/L-0.0350 g/L of disodium hydrogen phosphate,
1.8732-2.0120 g/L of sodium bicarbonate,
0.00058g/L to 0.00062g/L of zinc sulfate heptahydrate,
Linoleic acid 0.00018 g/L-0.00022 g/L,
0.00028 g/L-0.00032 g/L of sodium selenite,
0.0001-0.0015 g/L hydrocortisone,
0.0003 g/L-0.0004 g/L of lipoic acid,
0.0633 g/L-0.0680 g/L of sodium pyruvate.
6. A transfection method for 293 cells according to claim 5, wherein the concentration of L-tyrosine in the transfection medium is between 0.198g/L and 0.208 g/L.
7. A transfection method for 293 cells according to claim 5, wherein the concentration of nicotinamide in the transfection medium is between 0.0015g/L and 0.0016 g/L.
8. The method of transfecting a 293 cell according to claim 5, wherein the magnesium sulfate is present in the transfection medium at a concentration of 0.026g/L to 0.036 g/L.
9. A transfection method for 293 cells according to claim 5, wherein the concentration of hydrocortisone in the transfection medium is between 0.0009g/L and 0.0010 g/L.
10. The method of transfecting a 293 cell according to claim 5, wherein the transfection medium further comprises glutamine at a concentration of 0.4g/L to 0.5 g/L.
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