CN111979190A - Culture medium for transfecting T cells and application thereof - Google Patents
Culture medium for transfecting T cells and application thereof Download PDFInfo
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- CN111979190A CN111979190A CN201910432914.8A CN201910432914A CN111979190A CN 111979190 A CN111979190 A CN 111979190A CN 201910432914 A CN201910432914 A CN 201910432914A CN 111979190 A CN111979190 A CN 111979190A
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/10—Metals; Metal chelators
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Abstract
The invention discloses a culture medium for transfecting T cells, which comprises the following components: dioleoyl lecithin, dioleoyl phosphatidyl serine sodium, cholesterol and divalent calcium salt, wherein the divalent calcium salt is CaCl2The molar ratio of dioleoyl lecithin, dioleoyl phosphatidyl serine sodium and cholesterol is 4:3: 3. The transfection efficiency of the invention to acute T lymphocyte leukemia cell lines and mouse primary T cells is as high as 65%, which is far higher than other commercialized liposome transfection reagents.
Description
Technical Field
The invention belongs to the technical field of immune cell transfection, and particularly relates to a culture medium for transfecting T cells and application thereof.
Background
Immune cells are difficult to be affected by DNA or RNA delivered by conventional liposome materials due to their specific functions and properties. Either plasmid overexpression, CRISPR knockout, or siRNA silencing, lipofection is not effective in immune cells and transfection efficiency is often less than 20%. In the prior art, commonly used lipofection products, such as Lipofectamine3000, Polyjet, FugeneHD, and the like, cannot effectively transfect immune cells, such as macrophages, T cells, DC cells, and the like without exception.
The effective immune cell transfection methods in the prior art mainly comprise a virus transfection method and an electroporation transfection method. Viral transfection mainly employs lentiviruses, adenoviruses, adeno-associated viruses and the like for nucleic acid delivery. The virus transfection method has higher transfection efficiency on immune cells. According to different types of immune cells, the transfection efficiency of the virus transfection method on the immune cells can reach 70-95%. The current state-of-the-art CAR-T therapy is the in vitro genetic engineering of T cells isolated from patients by lentiviral transfection. However, the preparation of viruses is complicated by numerous steps and procedures, and is accompanied by difficult safety hazards (viruses can infect laboratory staff during the preparation process). The electroporation transfection method needs an electroporator and is simple and convenient to operate. However, electroporation, as its name implies, requires the perforation of the cell surface, which results in a significantly reduced survival rate of the cells after electroporation, and a lower transfection efficiency than viral transfection.
Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a liposome culture medium formula with high transfection efficiency on immune cells.
The technical problem to be solved by the invention is to provide the application of the liposome culture medium formula with high transfection efficiency on immune cells.
In order to solve the technical problems, the invention adopts the following technical scheme:
a medium for transfecting T cells comprising the following components: dioleoyl lecithin, dioleoyl phosphatidyl serine sodium, cholesterol, and divalent calcium salt.
Dioleoyl phosphatidylserine sodium has an immunosuppressive effect (immnuo-suppression), and can inhibit the activity of immune cells during transfection, so that the phenomenon that a transfection complex is degraded by the immune cells too early to play a role is avoided.
Dioleoyl lecithin (DOPC, 1, 2-Dioleoyl-sn-glycerol-3-phosphatidylcholine) has the following structure:
dioleoyl phosphatidylserine sodium (DOPS, 1, 2-dioleoyl-sn-glycerol-3-phospho-L-serine sodium salt) has the following structure:
cholesterol (cholestrol), whose structure is as follows:
wherein the divalent calcium salt is CaCl2。
Wherein the molar ratio of dioleoyl lecithin, dioleoyl phosphatidyl serine sodium and cholesterol is 4:3: 3.
Wherein the content of the divalent calcium salt is 2 mM.
Wherein the concentration of dioleoyl lecithin is 4.8nM, the concentration of dioleoyl phosphatidylserine sodium is 3.6mM, and the concentration of cholesterol is 3.6 nM.
Wherein the formulation comprises nucleic acid.
Wherein the nucleic acid is RNA or DNA;
when the nucleic acid is RNA, the RNA content is 20-160 nM, preferably 40 nM;
when the nucleic acid is DNA, the content of the DNA is 40-320 nM, preferably 80 nM.
Wherein, the formula comprises a cell culture medium which is a DMEM culture medium or an RPMI-1640 culture medium.
A method of liposome-mediated cell transfection comprising the steps of:
preparing a liposome transfection mother liquor containing dioleoyl lecithin, dioleoyl phosphatidylserine sodium and cholesterol, wherein the molar ratio of the dioleoyl lecithin to the dioleoyl phosphatidylserine sodium to the cholesterol is 4:3: 3; the transfection mother liquor liposome formula is named as Transimmunax (Ti for short);
(2) adding liposome transfection mother liquor, CaCl2 solution and nucleic acid to be transfected into a cell culture medium to obtain a transfection culture medium;
the concentration of dioleoyl lecithin in the transfection medium was 4.8nM, the concentration of dioleoyl phosphatidylserine sodium was 3.6mM, and the concentration of cholesterol was 3.6 nM;
the cell culture medium is DMEM or RPMI-1640 culture medium;
the concentration of CaCl2 solution in the transfection medium is 2 mM;
the nucleic acid to be transfected is RNA or DNA, the final concentration of the RNA is 20-160 nM, and the final concentration of the DNA is 40-320 nM;
(3) Removing a complete culture medium of a cell to be transfected, adding a transfection culture medium, and culturing for 24h, wherein the cell to be transfected is a T cell or a primary T cell;
(4) and removing the transfection medium, adding the complete medium, and continuously culturing for 24h to obtain the cells successfully transfected.
The liposome formulation of the transfection T cell is applied to acute T lymphocyte leukemia cell, primary T cell, A549 cell, H1299 cell, HeLa cell and SW480 cell transfection.
Has the advantages that:
1. the transfection efficiency of the liposome transfection formula (Ti formula) and the method to the T cell line and the primary T cell is far higher than that of other commercialized liposome transfection reagents. The transfection efficiency of the transfection liposome of the invention to acute T lymphocyte leukemia cell line and mouse primary T cell is as high as 65%, which is much higher than other commercial liposome transfection reagent (< 20%). The transfection efficiency of the transfection liposome of the invention in cancer cell lines A549, H1299, HeLa and SW480 is over 85%.
2. The lipofection formula and the lipofection method of the invention have lower cytotoxicity to T cell lines and primary T cells than other commercialized lipofection reagents.
3. The liposome transfection formulation and method of the present invention are less cytotoxic to T cell lines and primary T cells than electroporation.
4. The liposome transfection method of the invention is simpler to implement compared with the preparation of lentiviruses.
5. The biological safety of the liposome formulation of the invention is much higher than that of lentiviruses.
6. The cost of the liposome formulation of the present invention is lower than that of lentiviruses. .
Drawings
FIG. 1 fluorescence image of transfected GFP plasmid of Jurkat cells.
FIG. 2 open-field graph of transfection of Jurkat cells with GFP plasmid.
FIG. 3 fluorescence map of mouse primary T cells transfected with GFP plasmid.
FIG. 4 open-field graph of transfection of mouse primary T cells with GFP plasmid.
FIG. 5 flow chart of Jurkat cells transfected with GFP plasmid 48 h.
FIG. 6 flow chart of mouse primary T cells transfected with GFP plasmid 48 h.
FIG. 7 Gene knockdown efficiency map, qPCR detection by Ti and Lipofectamine3000 method by siRNA knockdown FADD and RAF1 gene expression in Jurkat cells, Scaramble control as negative control siRNA.
FIG. 8 is a gene knockdown efficiency graph, wherein expression of FADD and RAF1 genes in murine primary T cells was reduced by siRNA knockdown by Ti and Lipofectamine3000 in qPCR detection, and Scaramble control was negative control siRNA.
FIG. 9 is a Western blot detection result chart.
Detailed Description
Example 1: a method for preparing liposome transfection mother liquor.
(1) Mixing 4.65mg of DOPC, 3.60mg of DOPS sodium salt and 1.72mg of cholesterol powder, and dissolving in 10mL of chloroform to prepare 10mL of chloroform solution;
(2) Placing 10mL of chloroform solution obtained in the step (1) in a rotary evaporator for preparation for 4 hours, and evaporating a chloroform solvent;
(3) adding 2.5mL of PBS buffer solution into the dried round-bottom flask, and hydrating the lipid membrane;
(4) transferring the hydrated liposome suspension into a 5mL centrifuge tube, and placing the centrifuge tube at 4 ℃ for overnight shaking;
(5) treating the liposome suspension for 20 minutes by using an ultrasonic disruptor;
(6) the liposome solution was filtered using a 0.22M filter to obtain a concentration of approximately 6mM DOPS/DOPC/Chol liposome transfection stock.
(7) CaCl with the preparation concentration of 1M2The solution of (1).
Example 2: liposome-mediated cell transfection methods.
(1) mu.L of the lipofection mother liquor is added into 50 mu.L of RPMI-1640 culture medium;
(2) add 1. mu.L of LCaCl to another 50. mu.L of RPMI-1640 medium2And nucleic acid to be transfected, wherein the nucleic acid to be transfected is plasmid or siRNA (final transfection concentration of the nucleic acid is 80nM DNA and 40nM RNA);
(3) the two solutions were mixed to obtain 100. mu.L of transfected Master Mix. Standing at room temperature for 10 minutes;
(4) centrifuging to remove the complete medium of Jurkat cells in a 24-well plate, adding 100. mu.L of transfection Master mix, and centrifuging at 50g for 20 minutes at room temperature;
(5) taking out the centrifuged cell suspension, adding the cell suspension into a 24-pore plate, adding 400 mu L of complete culture medium, and culturing for 24 h;
(6) The medium containing the transfection mixture was removed by centrifugation and replaced with new complete medium. Continuously culturing for 24 h;
(7) the transfection effect can be detected by fluorescence, qPCR and Western blot.
Example 3: transfection of overexpression plasmids in Jurkat cells and mouse primary T cells.
pcDNA3.1-GFP plasmid was transfected into Jurkat and mouse primary T cells as described in example 2. Fluorescence of Jurkat and mouse primary T cells 48h after plasmid transfection is shown in FIGS. 1-4.
The Jurkat and the mouse primary T cells after GFP plasmid transfection for 48 hours are detected by a flow cytometer, the fluorescence rate of the Jurkat cells is about 76.9 +/-2.7 percent, and the fluorescence green of the mouse primary T cells is about 51.9 +/-1.9 percent.
Comparative example 1: jurkat and mouse primary T cells were transfected by Lipofectamine 3000.
(1) mu.L of Lipofectamine3000 transfection mother liquor was added to 50. mu.L of RPMI-1640 medium;
(2) add 1. mu.L of 40. mu.M pcDNA3.1-GFP plasmid to another 50. mu.L RPMI-1640 medium;
(3) mixing the two solutions to obtain 100. mu.L of transfected Master Mix, and standing at room temperature for 5 minutes;
(4) centrifuging to remove the complete culture medium of Jurkat cells or mouse primary T cells in a 24-well plate, adding 100. mu.L of transfection Master mix, and centrifuging at 50g for 20 minutes at room temperature;
(5) Taking out the centrifuged cell suspension, adding the cell suspension into a 24-pore plate, adding 400uL of complete culture medium, and culturing for 24 h;
(6) the medium containing the transfection mixture was removed by centrifugation and replaced with new complete medium. The culture was continued for 24 h.
The fluorescence rate of Jurkat and mouse primary T cells after 48h of transfection of Jurkat and mouse primary T cells by Lipofectamine3000 detected by a flow cytometer is 4.7 +/-0.6%.
Example 4: transfection of siRNA knockdown genes in Jurkat cells and mouse primary T cells using Ti transfection.
FADD and RAF1 genes of Jurkat cells were knocked down by siRNA using Ti transfection and Lipofectamine3000, respectively, by the methods in examples 2 and 3. After 48h, the qPCR assay results are shown in FIG. 7.
As can be seen from FIG. 7, the siRNA transfection of Jurkat cells with Ti resulted in FADD knockdown efficiencies of about 69.9. + -. 1.5% and RAF1 knockdown efficiencies of about 75.7. + -. 0.9%; the FADD knockdown efficiency was about 15.5. + -. 2.4% and the RAF1 knockdown efficiency was about 18.3. + -. 2.0% using Lipofectamine3000 transfection of siRNA.
The results of our qPCR assay for reducing FADD and RAF1 genes (siFADD: 5'-GAAGACCUGUGUGCAGCAUTT-3', siRAF 1: 5'-AGGAUCUGUGAGUUUGCCAUC-3') of murine primary T cells by siRNA knockdown using Ti transfection and Lipofectamine3000 are shown in FIG. 8.
As shown in FIGS. 7-8, siRNA transfection of primary mouse T cells with Ti resulted in FADD knockdown efficiencies of about 64.3. + -. 0.7% and RAF1 knockdown efficiencies of about 82.7. + -. 0.8%; the FADD knockdown efficiency was about 9.5. + -. 1.2% and the RAF1 knockdown efficiency was about 11.3. + -. 1.1% when Lipofectamine3000 was used to transfect siRNA.
We knocked down the FADD gene of Jurkat and mouse primary T cells by siRNA by Ti transfection and Lipofectamine 3000. After 48h, the Western blot detection result is shown in FIG. 9. Compared with Lipofectamine3000, the knockout efficiency of Ti (Ti + siRNA) at protein level is significantly higher than that of Lipofectamine3000(Lipo3000 + siRNA), while scramblecontrol (Ti + sNC and Lipo3000+ sNC) has no significant knockout effect on the target protein (sNC is siRNA negative control with no effect).
Claims (10)
1. A medium for transfecting T cells comprising the following components: dioleoyl lecithin, dioleoyl phosphatidyl serine sodium, cholesterol, and divalent calcium salt.
2. The medium of claim 1, wherein the divalent calcium salt is CaCl2。
3. A medium for transfecting a T-cell according to claim 1, wherein said dioleoyl lecithin, dioleoyl phosphatidylserine sodium and cholesterol are present in a molar ratio of 4:3: 3.
4. A medium for the transfection of T-cells according to claim 1 or 2 characterized in that the content of divalent calcium salt is 2 mM.
5. A medium for transfecting a T-cell according to claim 1 or 2, wherein said dioleoyl lecithin is at a concentration of 4.8nM, dioleoyl phosphatidylserine sodium is at a concentration of 3.6nM, and cholesterol is at a concentration of 3.6 nM.
6. The medium of claim 1, comprising a nucleic acid.
7. A medium for transfecting a T-cell according to claim 6, wherein said nucleic acid is RNA or DNA;
when the nucleic acid is RNA, the RNA content is 20-160 nM;
when the nucleic acid is DNA, the content of the DNA is 40-320 nM.
8. A medium for the transfection of T cells according to claim 1 characterized in that the culture comprises a cell culture medium, said cell culture medium being DMEM medium or RPMI-1640 medium.
9. A method of liposome-mediated cell transfection, comprising the steps of:
(1) preparing a liposome transfection mother liquor containing dioleoyl lecithin, dioleoyl phosphatidylserine sodium and cholesterol, wherein the molar ratio of the dioleoyl lecithin to the dioleoyl phosphatidylserine sodium to the cholesterol is 4:3: 3;
(2) Adding liposome transfection mother liquor, CaCl2 solution and nucleic acid to be transfected into a cell culture medium to obtain a transfection culture medium;
the concentration of dioleoyl lecithin in the transfection medium was 4.8nM, the concentration of dioleoyl phosphatidylserine sodium was 3.6mM, and the concentration of cholesterol was 3.6 nM;
the cell culture medium is a DMEM culture medium or an RPMI-1640 culture medium;
the concentration of CaCl2 solution in the transfection medium is 2 mM;
the nucleic acid to be transfected is RNA or DNA, the final concentration of the RNA is 20-160 nM, and the final concentration of the DNA is 40-320 nM;
(3) removing a complete culture medium of a cell to be transfected, adding a transfection culture medium, and culturing for 24h, wherein the cell to be transfected is a T cell or a primary T cell;
(4) and removing the transfection medium, adding the complete medium, and continuously culturing for 24h to obtain the cells successfully transfected.
10. Use of a medium according to any one of claims 1 to 8 for the transfection of T cells in the transfection of acute T lymphoblastic leukemia cells, primary T cells, a549 cells, H1299 cells, HeLa cells, SW480 cells.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001080900A2 (en) * | 2000-04-20 | 2001-11-01 | The University Of British Columbia | Enhanced stabilised plasmid-lipid particle-mediated transfection using endosomal membrane |
| CN107095799A (en) * | 2017-06-21 | 2017-08-29 | 广州市禾基生物科技有限公司 | Complex liposome and its preparation method and application |
| WO2017223085A2 (en) * | 2016-06-20 | 2017-12-28 | The Regents Of The University Of Michigan | Compositions and methods for delivery of biomacromolecule agents |
| CN108136050A (en) * | 2015-08-28 | 2018-06-08 | 分子传递有限公司 | Transfect complex compound and its application method |
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- 2019-05-23 CN CN201910432914.8A patent/CN111979190A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001080900A2 (en) * | 2000-04-20 | 2001-11-01 | The University Of British Columbia | Enhanced stabilised plasmid-lipid particle-mediated transfection using endosomal membrane |
| CN108136050A (en) * | 2015-08-28 | 2018-06-08 | 分子传递有限公司 | Transfect complex compound and its application method |
| WO2017223085A2 (en) * | 2016-06-20 | 2017-12-28 | The Regents Of The University Of Michigan | Compositions and methods for delivery of biomacromolecule agents |
| CN107095799A (en) * | 2017-06-21 | 2017-08-29 | 广州市禾基生物科技有限公司 | Complex liposome and its preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| LAI 等: "Highly efficient siRNA transfection in macrophages using apoptotic body-mimic Ca-PS lipopolyplex", 《INTERNATIONAL JOURNAL OF NANOMEDICINE》 * |
| TARAFDAR等: "Phosphatidylserine inhibits and calcium promotes model membrane fusion", 《BIOPHYSICAL JOURNAL》 * |
| 焦姣 等: "脂质体药物传递系统的50年发展历程概述", 《沈阳药科大学学报》 * |
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