CN111808880A - Electrotransfection buffer solution and application thereof - Google Patents
Electrotransfection buffer solution and application thereof Download PDFInfo
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Abstract
The invention discloses an electrotransfection buffer solution and application thereof, wherein the electrotransfection buffer solution comprises an Opti-MEM culture medium; the application of the electrotransfection buffer solution is that the electrotransfection buffer solution is used for resuspending cells to be electrotransfected, then an exogenous gene vector is added, and an electrotransfection system is obtained through mixing; the electrotransfection buffer solution has definite components and does not contain components of animal sources or allogeneic sources, so that the safety and the compliance of the cells for electrotransfection are improved; the electrotransfection buffer solution is applied to the electrotransfection process of cells, so that the electrotransfection of the cells is realized, and the stability and the success rate of the electrotransfection process are improved.
Description
Technical Field
The invention relates to an electrotransfection buffer solution and application thereof, belonging to the technical field of biology.
Background
Electrotransfection is one of the most commonly used techniques in molecular biology, and by providing transient local voltage, electroporation on cell membranes and electrophoresis action during the transportation of charged molecules are formed, the cell plasma membranes are permeated, drugs and gene substances can be transferred into cells cultured in vitro, and the direct cell transportation of the drugs and the editing of cell functions are realized. Thereby realizing clinical application. The electrotransfection process needs to provide a proper electrotransfection buffer system to maintain the cell sensitive conditions such as ion concentration, osmotic pressure, pH and the like of the environment where the cells are in the electrotransfection process. However, the main components of the current commercial electrotransformation liquid are inorganic salt components, the components are not clear enough and not completely disclosed due to confidentiality reasons, and whether the components harmful to human bodies or cells exist in the subsequent clinical application process cannot be scientifically judged, so that the potential safety hazard to cell treatment is huge.
Disclosure of Invention
In order to overcome the defects of the prior art, the first object of the invention is to provide an electrotransfection buffer solution which has definite components and does not contain components of animal origin or allogeneic origin, and the safety and the compliance of the electrotransfection of cells are improved.
The second purpose of the present invention is to provide an application of the above-mentioned electrotransfection buffer solution, in order to apply the electrotransfection buffer solution to the process of electrotransfection of cells, to realize the electrotransfection of cells, and to improve the stability and success rate of the electrotransfection process.
The first purpose of the invention can be achieved by adopting the following technical scheme: an electrotransfection buffer comprising Opti-MEM medium.
The second purpose of the invention can be achieved by adopting the following technical scheme: use of an electrotransfection buffer as described above in a process for electrotransfection of cells.
Further, the electrotransfection buffer solution is used for resuspending the cells to be electrotransfected, then the exogenous gene vector is added, and the electrotransfection system is obtained by mixing.
Further, the foreign gene vector is mRNA.
Furthermore, in the electrotransfection system, the concentration of the exogenous gene vector is 1-5pg/cell per cell to be electrotransfected; the concentration of cells to be electroporated is 50-100 nM.
Further, the process of cell electrotransfection is as follows: and adding the electrotransfection system into the middle groove of the electrotransfection cup, placing the electrotransfection cup into the groove of the electrotransformation machine electrode, and starting the electrotransfection machine to electrotransfect the cells.
Furthermore, cells were electrotransferred using a HARVARD APPATUS brand BTX Agile Pulse MAX electrotransferrer with the parameters of 1200V voltage, 0.1ms length of electric Pulse, 0.2ms interval, 1 time of electric Pulse.
Further, after the electrotransfection is completed, the cell suspension in the electrotransfer cup is taken out, added into an expression culture medium and then put into CO2Culturing in an incubator for 20 +/-4H.
Further, CO2The culture conditions in the incubator are 37 + -1 deg.C of temperature and CO2The concentration is 5 +/-0.5%.
Further, the expression medium is AIM-V CTS serum-free cell culture medium containing 5-10 vt% Knockout SR serum substitute and 50-200IU/mL recombinant interleukin rIL-2.
The formulation design principle of the invention is as follows:
in the industry, although the Opti-MEM culture medium is in the prior art, the Opti-MEM culture medium is used in cell culture in a conventional way, but the invention application research considers that the Opti-MEM culture medium can be used as an electrotransfection buffer solution to be applied to the process of cell electrotransfection, and the electrotransfection buffer solution has definite components and does not contain components of animal sources or allogeneic sources, so that the transfected cells have stable growth, and the invention has better controllability for the research of the subsequent application of the cells, and avoids the safety and compliance risks caused by introducing the electrotransfection buffer solution with unclear components when the cells are subjected to electrotransfection operation
Compared with the prior art, the invention has the beneficial effects that:
1. although the Opti-MEM culture medium in the electrotransfection buffer solution is conventional, the Opti-MEM culture medium is conventionally used in cell culture, but the research of the invention application of the invention considers that the Opti-MEM culture medium can be used as the electrotransfection buffer solution to be applied to the process of the cell electrotransfection, belongs to the development of new application, and is different from the conventional cell culture function, because the components of the electrotransfection buffer solution are definite and do not contain components of animal sources or allogeneic sources, the cells after transfection grow stably;
2. the electrotransfection buffer solution has definite components, so that the subsequent application research of the successfully transferred cells has better controllability, and the safety and compliance risks caused by introducing the electrotransfection buffer solution with unclear components when the cells are subjected to electrotransfection operation are avoided;
3. the application of the electrotransfection buffer solution is different from the conventional cell culture function, provides a good electrotransfection environment for cells in the electrotransfection process, and improves the success rate of the electrotransfection.
Drawings
FIGS. 1-4 are schematic representations of a flow assay for cell phenotype;
FIG. 5 is a bar graph comparing the expression rate of exogenous mRNA on CD8+ T cells;
FIG. 6 is a bar graph comparing the total number of viable cells;
FIG. 7 is a bar graph comparing cell viability;
FIG. 8 is a bar graph comparing the expression rates of intracellular INF-gamma and TNF-a cells.
Detailed Description
The invention will be further described with reference to the accompanying drawings and the detailed description below:
an electrotransfection buffer solution is an Opti-MEM culture medium; the Opti-MEM Medium is Gibco brand CTS Opti-MEM Medium.
The Opti-MEM culture medium is used as an electrotransfection buffer solution and is used for the process of electrotransfection of cells, and the specific steps are as follows:
1. preparing an expression culture medium: preparation-AIM-V CTS Serum-free cell culture medium of 510 vt% Knockout SR Serum Substitute (SR) and 50-200IU/mL recombinant interleukin rIL-2; the expression culture medium is used for culturing the cells after the electrotransfection and rinsing the electric rotor, and the expression culture medium for culturing the cells is transferred to each hole of the 12-hole plate; the expression medium can sufficiently support the growth of cells after electrotransfection and the expression of exogenous gene sequences;
2. preparing an electrotransfection system: centrifuging cell suspension to be subjected to electrotransfection, discarding the supernatant, and then resuspending the cells to be subjected to electrotransfection with an electrotransfection buffer solution to ensure that the concentration of the cells to be subjected to electrotransfection is 50-100nM, adding an exogenous gene vector to each cell to be subjected to electrotransfection with the concentration of 1-5pg/cell, and lightly blowing, sucking and uniformly mixing to obtain an electrotransfection system; wherein the exogenous gene vector includes but is not limited to nucleotide, DNA and RNA, protein, saccharide, dye, virus particle, etc.;
3. electrotransfection step: adding the electrotransfection system into the middle groove of an electrotransfer cup (goods number: 45-0126), placing the electrotransfer cup into the groove of an electrotransfer instrument electrode, and electrotransfecting cells by using a HARVARD APPATUS brand BTX Agile Pulse MAX electrotransfer instrument; adjusting the electric conversion parameters of the electric conversion instrument to be 1200V of voltage, 0.1ms of electric pulse length, 0.2ms of interval and 1 time of electric pulse;
4. expression culture: after the electrotransfection is finished, taking out the cell suspension in the electrotransfer cup, adding the cell suspension into an expression culture medium of a 12-hole plate, and then adding CO2Culturing 20 +/-4H and CO in an incubator2The culture conditions are 37 + -1 deg.C of temperature, CO2The concentration was 5. + -. 0.5%, and the cells were obtained after completion of the electrotransfection.
The application of the invention uses a new electrotransfection buffer solution, and in the application process, the electrotransfection system and the electrotransfection process parameters need to be correspondingly improved; through research, a specific electrotransfection buffer system is set, so that cells have good ion concentration, osmotic pressure and pH value in the electrotransfection process; the electrotransformation instrument parameters of electrotransfection are also set according to the used electrotransfection buffer solution, and the operability of the electrotransfection buffer solution is proved.
Example 1:
an electrotransfection buffer solution is an Opti-MEM culture medium; the Opti-MEM Medium is Gibco brand CTS Opti-MEM Medium.
The Opti-MEM culture medium is used as an electrotransfection buffer solution and is used for the process of electrotransfection of cells, and the specific steps are as follows:
1. preparing an expression culture medium: preparing an AIM-V CTS Serum-free cell culture medium of 10vt percent Knockout SR Serum Substitute (SR) and 100IU/mL recombinant interleukin rIL-2; the expression culture medium is used for culturing the cells after the electrotransfection and rinsing the electric rotor, and the expression culture medium for culturing the cells is transferred to each hole of the 12-hole plate; the expression medium can sufficiently support the growth of cells after electrotransfection and the expression of exogenous gene sequences;
2. preparing an electrotransfection system: centrifuging cell suspension to be subjected to electrotransfection, discarding supernatant, resuspending the cells to be subjected to electrotransfection with 200 mu L of electrotransfection buffer solution to ensure that the concentration of the cells to be subjected to electrotransfection is 80-90nM, adding exogenous gene vector mRNA (messenger ribonucleic acid) with the concentration of 1-5pg/cell into each cell to be subjected to electrotransfection, and lightly blowing, sucking and uniformly mixing to obtain an electrotransfection system;
3. electrotransfection step: adding the electrotransfection system into the middle groove of an electrotransfer cup (goods number: 45-0126), placing the electrotransfer cup into the groove of an electrotransfer instrument electrode, and electrotransfecting cells by using a HARVARD APPATUS brand BTX Agile Pulse MAX electrotransfer instrument; adjusting the electric conversion parameters of the electric conversion instrument to be 1200V of voltage, 0.1ms of electric pulse length, 0.2ms of interval and 1 time of electric pulse;
4. expression culture: electrotransfection is finishedAfter the completion, the cell suspension in the cuvette was taken out, added to the expression medium in a 12-well plate, and then placed in CO2Culturing 20 +/-4H and CO in an incubator2The culture conditions are 37 + -1 deg.C of temperature, CO2The concentration was 5. + -. 0.5%, and the cells were obtained after completion of the electrotransfection.
Comparative examples 1 to 2:
comparative example 1 is the use of HARVARD APPATUS brand T4 buffer as the electrotransfection buffer; comparative example 2 is the use of HARVARD APPATUS brand T buffer as the electrotransfection buffer; the expression medium, the electrotransfection instrument and the operation steps are the same as those in the example 1; wherein the electrotransfer parameter of the electrotransfer instrument of comparative example 1 is voltage 1200V, electric pulse length 0.1ms, interval 100ms, electric pulse 1 time; the electrotransfer instrument of comparative example 2 has electrotransfer parameters of 130V voltage, 0.2ms electric pulse length, 2ms interval and 4 electric pulses
The cells cultured in three groups, example 1 and comparative examples 1-2, were tested:
1. flow cytometry to detect cell specific phenotypes: the expression efficiency of exogenous mRNA on CD8+ T cells was analyzed by double positive values of CD8+ TCRV beta + (%) and shown in FIGS. 1-4, wherein FIG. 1 is a diagram of non-electrotransfected negative control cells, and FIGS. 2-4 represent example 1 and comparative examples 1-2, respectively; FIG. 5 is a bar graph comparing the efficiency of the bit representations;
2. the total number of the living cells of each group is detected by a microscopic counting method: the results are shown in FIG. 6;
3. the microscopic counting method is used for detecting the cell survival rate of each group: the results are shown in FIG. 7;
4. the effect target cell co-culture method is used for detecting the secretion functions of cytokine INF-gamma and TNF-a of effector cells (CD8+ TCRV beta +), after the cells after electrotransfection and HLA (human leukocyte common antigen) B cells after gene modification are incubated for 18h, the expression rates of intracellular INF-gamma and TNF-a cells are detected in a flow mode, and the results are shown in figure 8.
Various other changes and modifications to the above-described embodiments and concepts will become apparent to those skilled in the art from the above description, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Claims (10)
1. An electrotransfection buffer, characterized in that said electrotransfection buffer comprises an Opti-MEM medium.
2. Use of the electrotransfection buffer according to claim 1 in a cell electrotransfection process.
3. The use of the electrotransfection buffer according to claim 2, wherein the electrotransfection buffer is re-suspended in the cell to be transfected, then the exogenous gene vector is added and mixed to obtain the electrotransfection system.
4. The use of the electrotransfection buffer according to claim 3, wherein the foreign gene vector is mRNA.
5. The use of the electrotransfection buffer according to claim 3, wherein the concentration of the exogenous gene vector in the electrotransfection system is 1-5pg/cell per cell to be electrotransfected; the concentration of the cells to be electroporated is 50-100 nM.
6. The use of the electrotransfection buffer according to claim 3, wherein the cell electrotransfection process is: and adding the electrotransfection system into the middle groove of the electrotransfection cup, placing the electrotransfection cup into the groove of the electrotransformation machine electrode, and starting the electrotransfection machine to electrotransfect the cells.
7. The use of the electrotransfection buffer according to claim 6, wherein the cells are electrotransferred using a HARVAR D APPATUS brand BTX Agile Pulse MAX electrotransferrer with the parameters of 1200V voltage, 0.1ms electric Pulse length, 0.2ms interval and 1 electric Pulse.
8. The use of the electrotransfection buffer of claim 6, wherein after the electrotransfection is completed, the cell suspension in the electrotransfer cell is taken out and the expression medium is addedThen put in CO2Culturing in an incubator for 20 +/-4H.
9. Use of the electrotransfection buffer according to claim 8, wherein the CO is2The culture conditions in the incubator are 37 + -1 deg.C of temperature and CO2The concentration is 5 +/-0.5%.
10. Use of the electrotransfection buffer according to claim 8, wherein the expression medium is an A IM-V CTS serum-free cell culture medium containing 5-10 vt% Knockout SR serum replacement and 50-200IU/mL recombinant interleukin rIL-2.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528437A (en) * | 2021-07-07 | 2021-10-22 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Kit for enhancing gene editing efficiency and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105154472A (en) * | 2015-09-28 | 2015-12-16 | 重庆高圣生物医药有限责任公司 | Mammalian cell efficient electrotransfection buffer solution and preparation method thereof |
| CN106995783A (en) * | 2016-01-22 | 2017-08-01 | 上海交通大学 | Cell electrotransfection device and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154472A (en) * | 2015-09-28 | 2015-12-16 | 重庆高圣生物医药有限责任公司 | Mammalian cell efficient electrotransfection buffer solution and preparation method thereof |
| CN106995783A (en) * | 2016-01-22 | 2017-08-01 | 上海交通大学 | Cell electrotransfection device and its application |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528437A (en) * | 2021-07-07 | 2021-10-22 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Kit for enhancing gene editing efficiency and application thereof |
| CN113528437B (en) * | 2021-07-07 | 2023-08-25 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Kit for enhancing gene editing efficiency and application thereof |
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