CN114350595A - Lentiviral titer detection method for HEK293TS suspension cells - Google Patents
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0684—Cells of the urinary tract or kidneys
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Abstract
The invention discloses a method for detecting lentivirus titer of HEK293TS suspension cells, which comprises the following steps: adjusting the density of HEK293TS suspension cells and mixing; spreading a proper amount of HEK293TS suspension cells in a 24-well plate; pre-diluting the lentivirus solution by multiple times, adding a proper amount of pre-diluted virus solution into each hole of a 24-hole plate, and supplementing a proper amount of virus diluent to ensure that the volumes of the virus solutions in the holes are the same; using CO2Culturing HEK293TS cells by a shaking table, respectively culturing for 0h, 24h and 48h, respectively detecting the viable cell density and the cell viability after 0h and 24h, and carrying out flow detection on the titer of the virus after 48h of culture. The detection method of the invention has simple operation, short detection period, high detection flux and can avoidAvoids the problem of serum batch difference existing in the culture of HEK293T adherent cells; the virus diluent does not cause the reduction of the cell viability and the viable cell density of HEK293TS suspension cells; the invention adopts CO2The shaking table is used for culturing the virus, so that the cell survival rate, the viable cell density and the FACS infection titer are improved.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting lentivirus titer of HEK293TS suspension cells.
Background
HEK293T cells were widely used for lentiviral packaging and titer detection. The adherent HEK293T cells are inevitably different in serum batch due to the adherent HEK293T cell culture problem, and the adherent cells are lower in detection flux and long in period compared with suspension cells in virus titer detection, and need pancreatin digestion treatment, so that the operation is complicated. And the HEK293TS suspension cell is cultured in a serum-free manner, so that the problem of difference between serum batches does not exist, and the HEK293TS suspension cell has the advantages of high experimental throughput, shorter whole detection process period, no need of pancreatin treatment, simplicity in operation and the like when being used for detecting the virus titer. However, there is no report on the use of this method for the determination of lentiviral transduction titers.
Disclosure of Invention
The invention aims to provide a method for detecting the lentivirus titer of HEK293TS suspension cells, which can solve the problems that the method for detecting the lentivirus titer by using adherent HEK293T cells in the background art is low in flux, long in period, needs pancreatin treatment and is complex to operate.
In order to achieve the purpose, the invention is realized by the following technical scheme: a method for detecting the lentivirus titer of HEK293TS suspension cells comprises the following steps:
s1, adjusting the density of HEK293TS suspension cells and mixing uniformly;
s2, uniformly mixing, and paving a proper amount of HEK293TS suspension cells in a 24-pore plate;
s3, pre-diluting the lentivirus solution by multiple times, adding a proper amount of pre-diluted virus solution into each hole of a 24-hole plate, and then supplementing a proper amount of virus diluent, wherein the virus diluent comprises a CD01 culture medium or a DPBS buffer solution, so that the volumes of the virus solutions in the holes are the same;
s4, use of CO2Culturing HEK293TS cells in a 24-well plate by using a shaking table, respectively culturing for 0h, 24h and 48h, respectively detecting the viable cell density and the cell viability after 0h and 24h, and carrying out flow detection on the titer of the virus after 48h of culture.
As a further improvement of the method for detecting the lentivirus titer of the HEK293TS suspension cells, the cell density was adjusted to 4.5X 10 in step S15cells/mL-9.5×105cells/mL。
As a further improvement of the above method for detecting the lentivirus titer of the HEK293TS suspension cells, 1ml of HEK293TS suspension cells were added to each well of a 24-well plate in step S2.
As a further improvement of the method for detecting the lentivirus titer of the HEK293TS suspension cells, in step S3, different amounts of pre-diluted virus solution are added to each well, and then virus dilutions are added to make the volumes of the virus solutions in the wells the same.
The invention has the beneficial effects that: 1) the method for detecting the lentivirus titer of the HEK293TS suspension cells can detect the lentivirus titer of the HEK293TS suspension cells, can avoid the problem of difference between serum batches inevitably existing in adherent HEK293T cell culture, has short detection period, does not need pancreatin digestion treatment, and is simple to operate and high in detection flux; 2) according to the method for detecting the lentivirus titer of the HEK293TS suspension cells, the CD01 culture medium or the DPBS buffer solution is used as the virus diluent, so that the problem that the cell viability and the viable cell density of the suspension HEK293TS cells are reduced after the DMEM culture medium is added in the prior art can be solved, and therefore, the cell viability and the viable cell density of the suspension HEK293TS cells can be improved by adopting the CD01 culture medium; 3) in step S4 of the present invention, CO is used2The shaking table is used for culturing the virus, and CO is adopted in the prior art2Compared with the scheme of the incubator, the cell viability, the viable cell density and the FACS infection titer are obviously improved.
Drawings
FIG. 1 is a graph of comparative example 1 showing the comparison of mean lentiviral titers obtained using the prior art method for detecting lentiviral titers of HEK293T adherent cells with the method for detecting lentiviral titers of HEK293TS suspension cells of the present invention.
FIG. 2 is a graph showing comparative data of cell density measured in comparative example 2 using different dilutions.
FIG. 3 is comparative data of cell viability measured using different dilutions in comparative example 2.
FIG. 4 shows the use of CO in comparative example 32Incubator and CO2Comparative data on cell viability obtained with the shaker culture mode.
FIG. 5 shows the use of CO in comparative example 32Incubator and CO2Comparative data on cell density obtained in the shaker culture mode.
FIG. 6 shows the use of CO in comparative example 32Incubator and CO2And (4) comparison data of lentivirus titer detected by a shaking table culture mode.
FIG. 7 is a comparison of lentivirus titers obtained in comparative example 4 using HEK293TS suspension cells at different cell densities.
The values on the coordinate axes in FIGS. 1, 2, 5, 6, 7, e.g. 5.00E +05 for 5X 105In the same way, 2.50E +09 represents 2.5X 109The other numerical values correspond to the above-mentioned exemplary expressions.
Detailed Description
The technical solutions of the present invention are described clearly and completely by the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for detecting the lentivirus titer of HEK293TS suspension cells comprises the following steps:
s1, HEK293TS suspension cells were adjusted to 8X 10 cell density5cells/mL；
S2, uniformly mixing, and paving in a 24-hole plate according to 1mL per hole;
s3, pre-diluting the virus by 100 times, respectively adding 1, 10 and 100 mu L of pre-diluted virus into a 24-well plate, and correspondingly adding 99, 90 and 0 mu L of virus diluent into the corresponding well, wherein the virus diluent is a CD01 culture medium;
s4, putting the 24-hole plate into CO2And (3) shaking the platforms, culturing for 0h, 24h and 48h respectively, uniformly mixing, sampling and detecting the living cell density and the living rate of the cells, directly and uniformly mixing by using a discharging gun, and cleaning, incubating the antibody, cleaning and carrying out flow detection on the virus titer after culturing for 48h after sampling.
Example 2
A method for detecting the lentivirus titer of HEK293TS suspension cells comprises the following steps:
s1, HEK293TS suspension cells were adjusted to 4.5X 10 cell density5cells/mL；
S2, uniformly mixing, and paving in a 24-hole plate according to 1mL per hole;
s3, pre-diluting the virus by 10 times, adding 20, 6.67, 2.22 and 0.74 mu L of pre-diluted virus into a 24-well plate respectively, and correspondingly adding 80, 93.33, 97.78 and 99.26 mu L of virus diluent which is a CD01 culture medium;
s4, putting the 24-hole plate into CO2And (3) shaking the platforms, culturing for 0h, 24h and 48h respectively, uniformly mixing, sampling and detecting the living cell density and the living rate of the cells, directly and uniformly mixing by using a discharging gun, and cleaning, incubating the antibody, cleaning and carrying out flow detection on the virus titer after culturing for 48h after sampling.
Example 3
A method for detecting the lentivirus titer of HEK293TS suspension cells comprises the following steps:
s1, HEK293TS suspension cells were adjusted to 9.5X 10 cell density5cells/mL；
S2, uniformly mixing, and paving in a 24-hole plate according to 1mL per hole;
s3, pre-diluting the virus by 50 times, adding 20, 6.67, 2.22 and 0.74 mu L of pre-diluted virus into a 24-well plate respectively, and correspondingly adding 80, 93.33, 97.78 and 99.26 mu L of virus diluent which is DPBS buffer solution;
s4, putting the 24-hole plate into CO2Shaking table, culturing for 0h, 24h, 48h, mixing well, sampling, detecting viable cell density and viable rate of cells, and usingAnd directly and uniformly mixing the components in a gun, and after sampling, cleaning, incubating the antibody, cleaning, and performing flow detection on the virus titer after culturing for 48 hours.
Comparative example 1
Comparing the lentivirus titer detection method of the HEK293T adherent cells in the prior art with the lentivirus titer detection method of the HEK293TS suspension cells to perform a titer detection comparison test on the same lentiviruses, the specific scheme is as follows:
the method for detecting the lentivirus titer of the HEK293T adherent cells in the prior art specifically comprises the following steps: HEK293T adherent cells were adjusted to density 4E +05cells/mL, uniformly mixing, spreading on a 24-well plate according to 0.5mL per well, randomly taking 3 wells after 24 hours, counting, and taking the average value of 4.39 multiplied by 105cells, pre-diluting virus 10 times, adding pre-diluted virus 20, 6.67, 2.22, 0.74 μ L, respectively, adding virus diluent 80, 99.33, 97.78, 99.26 μ L, respectively, adding CO 01 medium, respectively2And (3) adding 0.5mL of fresh culture medium into each hole after 24 hours in an incubator, continuously culturing for 24 hours, removing the supernatant, adding 100 mu L of pancreatin for digestion, stopping digestion by using 400 mu L of fresh culture medium, uniformly mixing, sampling, washing, incubating antibodies, washing and carrying out flow detection.
The lentivirus titer detection method of HEK293TS suspension cells is to adjust the cell density of HEK293TS suspension cells to 1.5 multiplied by 105cells/mL, and spreading the mixture in a 24-hole plate according to 1mL per hole after uniformly mixing; pre-diluting lentivirus 10 times, adding pre-diluted lentivirus 20, 6.67, 2.22, and 0.74 μ L, respectively, adding virus diluent 80, 99.33, 97.78, and 99.26 μ L, respectively, adding CD01 medium, and adding CO2And performing shake culture for 48h, uniformly mixing, sampling, cleaning, incubating the antibody, cleaning and performing flow detection.
By comparing the detection processes, the method for detecting the lentivirus titer of the HEK293TS suspension cells is simple and convenient to operate, does not need pancreatin digestion treatment, and can improve the detection efficiency.
As can be seen from fig. 1, when the infection titer of the same virus is detected, when the lentiviral titer detection is performed by using the HEK293TS suspension cell of the present invention and the HEK293T adherent cell of the prior art, the significant analysis results of the lentivirus are not different, and the detection results are consistent, which proves that the accuracy of the lentiviral titer detection performed by using the HEK293TS suspension cell of the present invention is high, and the HEK293TS suspension cell can completely replace the HEK293T adherent cell for the lentiviral titer detection; meanwhile, compared with the results of detecting the lentivirus titer by using the HEK293TS suspension cells in the prior art, the result of detecting the lentivirus titer by using the HEK293T adherent cells is higher in value, and the detection sensitivity can be improved.
Comparative example 2
The effect of different virus diluents on the cell growth is contrasted, and the specific scheme is as follows:
the cell density of the suspension of HEK293TS was adjusted to 6X 105And cells/mL, uniformly mixing, paving the mixture on a 24-well plate according to 1mL of each well, adding different virus diluents with the diluent of 100 mu L into each well, respectively culturing for 0h, 24h and 48h, uniformly mixing, and sampling to detect the viable cell density and the viable rate of the cells. The virus diluent respectively selects a D10 culture medium, a CD01 culture medium, a DBPS buffer solution and a mixture of the three substances and a three-antibody, wherein the formula of the D10 culture medium is a DMEM culture medium +10% FBS, the CD01 culture medium is a Transpro CD01 culture medium, the D10 culture medium is a commercial culture medium and can be used for HEK293TS suspension culture and virus packaging, and the virus of the harvested virus supernatant exists in a CD01 culture medium. DBPS buffer is a commercial buffer that is included in the purification process after virus packaging or in the finished product. The three-antibody is a mixed solution (100 multiplied by three-antibody) of penicillin-streptomycin-gentamicin, the three-antibody has the function of preventing bacteria from being introduced in the virus purification process, so that HEK293TS is polluted in the virus detection process and the detection experiment fails, and the addition amount of the three-antibody is 1X of working concentration.
As can be seen from fig. 2 and 3, the use of DMEM medium in the virus dilution resulted in a decrease in the cell viability and viable cell density of the suspended HEK293TS cells. The virus diluent selects a CD01 culture medium and a DPBS buffer solution or adds three antibodies into the two culture media, has no influence on the growth of HEK293TS cells, and proves that both the CD01 culture medium and the DPBS buffer solution can be used as the virus diluent.
Comparative example 3
Comparison ofCO is used in step S42Incubator and CO2The culture mode of the shaking table has influence on the growth of HEK293TS suspension cells and virus titer detection, and the specific scheme is as follows:
adjusting the cell density of the suspended HEK293TS to 8E +05cells/mL, uniformly mixing, spreading on a 24-well plate according to 1mL per well, pre-diluting the virus by 100 times, respectively adding 1, 10 and 100 mu L of the pre-diluted virus, correspondingly adding 99, 90 and 0 mu L of virus diluent (culture medium CD 01) into CO respectively2Incubator and CO2Shaking table culturing for 0h, 24h and 48h, mixing uniformly, sampling, detecting the viable cell density and the viable rate of the cells, and performing flow detection on the virus titer for 48 h.
As can be seen from FIGS. 4-6, the HEK293TS suspension cells were treated with CO after virus addition2The shaking table culture mode is adopted, and the cell viability, the viable cell density and the FACS infection titer of HEK293TS suspension cells are all higher than those of the HEK293TS suspension cells2The culture mode of the incubator is higher.
Comparative example 4
In the comparison step S1, a comparison test of lentivirus titer detection is performed using HEK293TS suspension cells of different cell densities, and the specific scheme is as follows:
the cell density of HEK293TS suspension cells was adjusted to 4.5X 105cells/mL、5.5×105cells/mL、6.5×105cells/mL、7.5×105cells/mL、8.5×105cells/mL、9.5×105cells/mL, uniformly mixing, spreading on a 24-well plate according to 1mL per well, pre-diluting the virus by 10 times, respectively adding 20, 6.67, 2.22 and 0.74 mu L of the pre-diluted virus, correspondingly adding 80, 99.33, 97.78 and 99.26 mu L of virus diluent which is CD01 culture medium, respectively adding CO2And performing shake culture for 48h, uniformly mixing, sampling, cleaning, incubating the antibody, cleaning and performing flow detection.
As can be seen from FIG. 7, the cell density of HEK293TS suspension cells was 4.5X 105-9.5×105The virus titer detection results in the cell/mL range have no significant difference, and the cell density of HEK293TS suspension cells is proved to be 4.5 multiplied by 105-9.5×105The result of detecting the virus titer in the cell/mL range has stability.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A method for detecting the lentivirus titer of HEK293TS suspension cells is characterized in that: which comprises the following steps:
s1, adjusting the density of HEK293TS suspension cells and mixing uniformly;
s2, uniformly mixing, and paving a proper amount of HEK293TS suspension cells in a 24-pore plate;
s3, pre-diluting the lentivirus solution by multiple times, adding a proper amount of pre-diluted virus solution into each hole of a 24-hole plate, and then supplementing a proper amount of virus diluent, wherein the virus diluent comprises a CD01 culture medium or a DPBS buffer solution, so that the volumes of the virus solutions in the holes are the same;
s4, use of CO2Culturing HEK293TS cells in a 24-well plate by using a shaking table, respectively culturing for 0h, 24h and 48h, respectively detecting the viable cell density and the cell viability after 0h and 24h, and carrying out flow detection on the titer of the virus after 48h of culture.
2. The method for detecting the lentivirus titer of HEK293TS suspension cells of claim 1, wherein the method comprises the steps of: in step S1, the cell density was adjusted to 4.5X 105cells/mL-9.5×105cells/mL。
3. The method for detecting the lentivirus titer of HEK293TS suspension cells of claim 1, wherein the method comprises the steps of: in step S2, 1ml of HEK293TS suspension cells were added to each well of the 24-well plate.
4. The method for detecting the lentivirus titer of HEK293TS suspension cells of claim 1, wherein the method comprises the steps of: in step S3, pre-diluted virus solutions with different amounts are added to each well, and then the virus diluent is added to make the volumes of the virus solutions in the wells the same.
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