CN113122501A - Culture medium suitable for preparation of large-scale clinical-grade virus vector and application thereof - Google Patents
Culture medium suitable for preparation of large-scale clinical-grade virus vector and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological viruses, and particularly relates to a culture method for preparing lentiviruses by culturing a vacceneXpress culture medium. The method can effectively reduce BSA (bovine serum albumin) residues, can be used for preparing clinical-grade lentiviruses, can obviously improve the titer of the lentiviruses, is suitable for large-scale clinical-grade lentivirus production, does not need serum-free domestication of working cells when being replaced to a serum-free culture medium, is simple and convenient, and consumes short time.
Description
Technical Field
The invention belongs to the field of biological viruses, and particularly relates to a method for culturing a lentiviral vector containing a target gene by using a glutamine-containing VaccireXpress culture medium.
Background
The current clinical gene therapy vectors are mainly viral vectors, and the main viral vectors include adenovirus, oncolytic virus and retrovirus. Among them, retroviral vectors, commonly used including gamma retrovirus and lentivirus vectors, have been used for clinical studies for stable gene transfer into mammalian cells for nearly 30 years. The retrovirus can be efficiently integrated into the genome of a target cell and stably and persistently express a carried foreign gene.
Lentiviral vectors belong to one class of retroviruses, and have a broader host range and are capable of efficiently infecting non-periodic and post-mitotic cells compared to other retroviruses. Moreover, the lentivirus vector is a gene therapy vector modified based on human immunodeficiency virus I (HIV), and the lentivirus vector from HIV-1 can infect non-dividing cells and can be stably integrated in vivo because of being capable of bearing larger exogenous target gene segments, and the phenomenon of gene silencing is less. And for some cells which are difficult to infect, such as primary cells, stem cells, undifferentiated cells and the like, the lentiviral vector is used, so that the transduction efficiency of the target gene can be greatly improved, and the probability of integrating the target gene into the host cell genome is increased, thereby realizing long-term and stable expression of the target gene more conveniently and quickly. The lentivirus vector and the immune response are small, the safety is good, and the like, and the lentivirus vector is widely applied to clinical research, and particularly the 3 rd generation self-defective lentivirus vector has extremely high biological safety.
With the development of research from the basic stage to the clinical stage, the demand of lentiviruses is greatly increased, the conventional lentivirus production method is difficult to meet, and a high-quality lentivirus preparation scheme meeting clinical requirements in large-scale production is urgently needed. The conventional lentivirus preparation method needs to use a DMEM medium containing FBS for culture, and liquid change is carried out before plasmid transfection and after plasmid transfection respectively, because the culture medium containing FBS is used, the produced lentivirus feed liquid contains a large amount of BSA residues, clinical-grade lentiviruses have clear limitation on the BSA residues, and the removal capacity of the lentivirus purification on the BSA is limited, so that the BSA residues after the lentivirus purification are still high and cannot meet the clinical requirements; on the other hand, although some serum-free culture media are available on the market at present, no culture media specially used for lentivirus preparation exists, and most of the serum-free culture media have poor effect on lentivirus preparation, so that a culture medium and a culture scheme thereof which can be used for lentivirus preparation are urgently needed.
Disclosure of Invention
In view of this, it is an object of the present invention to provide a combination of two media which can act synergistically in the packaging of lentiviral vectors and in the preparation of lentiviruses.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a combination of media suitable for large scale clinical grade viral vector production, including conventional media and vaccenexpress media.
Further, the conventional medium in the medium is DMEM medium containing FBS in an amount which is conventional to those skilled in the art, such as DMEM medium containing 5% or 10% FBS.
Furthermore, in the culture medium combination, glutamine is added into the VaccieXpress culture medium, and the addition amount of the glutamine is the conventional amount of a person skilled in the art, such as 1% of the glutamine.
Preferably, in the combination of media, the vacuneexpress medium is packaged separately.
The lentivirus vector is a virus vector derived from human immunodeficiency virus-1, contains genetic information required by packaging, transfection and stable integration, and is a main component of a lentivirus vector system. Under the assistance of lentivirus packaging plasmid and cell line, the lentivirus vector carrying target gene (functional gene) is virus-packaged into virus particle with infectivity, and the target gene is expressed in cell or tissue by infecting cell or living tissue. In the whole process, the invention adopts the combined application of two culture media: the vaccine Xprees culture medium can be used for preparing lentivirus, and the infection capacity of the lentivirus can be obviously improved by using the culture medium, which is far higher than other serum-free culture media and conventional DMEM culture medium with 5% FBS. Thus the use of the vacceneXprees medium for lentivirus production enables higher virus yields than conventional production protocols.
The invention also aims to provide a virus with less bovine serum albumin residual quantity and a preparation method thereof, and a product obtained by the method is suitable for clinical application scenes.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the method for reducing the residual amount of bovine serum albumin in the virus particles by using the culture medium combination is used for culturing a cell line capable of producing the virus particles by using the Vaccinexpress culture medium.
Further, the cell line is a HEK293T cell. HEK293T cells were used as packaging cells for lentivirus, and were anchorage-dependent epithelioid cells, and the conventional culture medium was DMEM medium containing FBS. The adherent cells grow and proliferate to form a monolayer of cells through culture. Cells of equivalent function, such as E.coli strain DH5a, can also be used to amplify lentiviral vectors and helper packaging vector plasmids.
Further, the method specifically comprises the following steps:
(1) pretreatment of
Culturing a cell line which can generate virus particles and is to be transfected by the conventional culture medium, and preparing a packaging plasmid and a slow virus plasmid carrying a target gene into a calcium phosphate-DNA compound; removing device
(2) Transfected cells
Adding the calcium phosphate-DNA complex into the cell line which can generate the virus particles and is cultured by the conventional culture medium, and obtaining the cell line which can generate the virus particles after the cell line is transfected, wherein the cell line which can generate the virus particles comprises the slow virus vector of the target gene;
(3) collection of viruses
The cell line producing virus particles is cultured in the VaccireXpress medium, and the virus is collected. Clinical-grade virus vector particles carrying the target gene and having only one-time infection capacity but no replication capacity can be obtained from the cell line.
Further, the time for culturing in the said VaccieXpress medium is 40 to 48 hours after plasmid transfection.
Specifically, the lentiviral vector containing the target gene comprises one of Human Immunodeficiency Virus (HIV), Simian Immunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV), Equine Infectious Anemia Virus (EIAV), Bovine Immunodeficiency Virus (BIV) and visna-midie virus (VMV) lentiviral plasmids.
The invention also aims to provide a new application of the VaccieXpress culture medium, which provides a new idea for large-scale production of lentiviral vectors.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the use of said vacceneXpress medium as a medium for said cell line capable of producing viral particles, such as transfected packaging cells.
In the application, glutamine is added into the VaccireXpress culture medium.
The VaccieXpress medium is a serum-free medium developed for vaccine production by Hyclone. The cell lines validated during the development of the medium are mainly adherent cells and microcarriers thereof (such as Vero cells and Vero cell microcarriers), and are used for the expression of influenza (influenza), Zika (Zika), chikura (Polio), Dengue (Dengue) and Respiratory Syncytial Virus (RSV) vaccine-related viruses for vaccine preparation. No data is available to show that the slow virus preparation method has been applied to the slow virus preparation, and the promotion of the application of the slow virus preparation method is not seen on the website of Hyclone company. The conventional lentivirus preparation method needs to use a DMEM medium containing FBS for culture, and liquid change is carried out before plasmid transfection and after plasmid transfection respectively, because the culture medium containing FBS is used, the produced lentivirus feed liquid contains a large amount of BSA residues, clinical-grade lentiviruses have clear limitation on the BSA residues, and the removal capacity of the lentivirus purification on the BSA is limited, so that the BSA residues after the lentivirus purification are still high and cannot meet the clinical requirements; the method is innovatively applied to lentivirus preparation, can effectively reduce BSA (bovine serum albumin) residue, can obviously improve the titer of the lentivirus, and does not need serum-free domestication on working cells when the culture medium is replaced to a serum-free culture medium.
Therefore, it is a fourth object of the present invention to provide a composition which can produce a high titer of viruses.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a composition suitable for large scale clinical grade viral vector production, said composition comprising said vaccenexpress medium and said viral particle producing cell line. Equivalently, compositions suitable for large-scale clinical-grade viral vector production comprise, after a certain period of time, said vacceneXpress medium and viral particles produced by said viral particle-producing cell line.
Further, the cell line in the composition is HEK293T cell.
Further, the composition consists of said vacceneXpress medium, glutamine and said cell line capable of producing viral particles.
The fifth purpose of the invention is to provide a method for improving the interference efficiency of the lentiviral vector, which is simple to operate and suitable for large-scale production.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for improving the infection efficiency of a lentiviral vector comprises the steps of transfecting a cell line capable of producing viral particles (containing the lentiviral vector containing the target gene) with the lentiviral vector containing the target gene under the conventional culture medium condition, and culturing the cell line by using the VaccieXpress culture medium, wherein the supernatant is the viral particles produced by the cell line.
Further, in the method, the gene of interest comprises a chimeric antigen receptor nucleic acid sequence or a gene therapy-related nucleic acid sequence.
Further, in said method said vacuneexpress medium contains 1-5% by volume of said glutamine.
Further, the lentiviral vector containing the target gene includes a viral vector, a vector containing a viral accessory protein gene, a vector containing a packaging protein gene, and a vector containing the target gene. It is also understood to include packaging plasmids, envelope plasmids, helper protein plasmids and lentiviral plasmids carrying the gene of interest.
Further, the method comprises the steps of: (1) culturing cells to be transfected in a DMEM medium containing FBS, wherein the transfected cells do not contain the target gene; preparing the packaging plasmid and the lentivirus plasmid carrying the target gene into a calcium phosphate-DNA compound; (2) adding the calcium phosphate-DNA compound into the cells to be transfected, and culturing the cells by using the DMEM medium containing FBS to obtain a cell line capable of producing virus particles, wherein the cell line capable of producing virus particles carries a lentiviral vector of a target gene; (3) the virus particle-producing cell line was subjected to supernatant removal and continued to culture in glutamine-containing Vaccinenes medium.
The calcium phosphate-DNA complex is obtained by calcium phosphate transfection methods well known in the art; other known methods may be substituted.
Further, in the culture at the non-transferred stage of the cells, the following manner can be referred to: firstly, culturing target cells without the lentiviral vector containing the target gene by using a DMEM medium containing 10-13% FBS, and then, replacing the DMEM medium containing 5-8% FBS for culture; preferably: the cells to be transfected (packaging cells) were cultured in DMEM medium containing 10-13% FBS, and then in DMEM medium containing 5-8% FBS for 3-8 hours. The two-time culture is preferably carried out at 37-40 deg.C and 5-8% CO2Culturing in a carbon dioxide incubator.
Further, the lentiviral plasmid carrying the gene of interest in the step (2) comprises one of Human Immunodeficiency Virus (HIV), Simian Immunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV), Equine Infectious Anemia Virus (EIAV), Bovine Immunodeficiency Virus (BIV), visna-midie virus (VMV) lentiviral plasmids.
Further, the calcium phosphate transfection method specifically comprises the following steps: and adding sterilized ultrapure water into a mixture of the packaged plasmid, the lentiviral plasmid carrying the target gene and 2-3mol/L CaCl2 solution, uniformly mixing, slowly adding a 2 x HBS solution with the pH value of 6.9-7.1 into the mixture, slowly dripping while uniformly mixing by oscillation, standing and incubating for 5-10 minutes after uniform mixing to obtain the calcium phosphate-DNA compound.
Further, the culture time using the said vaccineXpress medium was: 40-48 hours after plasmid transfer.
Further, the vacceneXpress medium can be replaced immediately without serum-free acclimation of the virus particle-producing cell line in the medium.
The invention has the beneficial effects that:
(1) the culture medium and the culture method provided by the invention can effectively reduce BSA (bovine serum albumin) residues and can be used for preparing clinical-grade lentiviral vectors;
(2) the culture medium and the culture method provided by the invention can obviously improve the titer of the lentivirus, and are suitable for large-scale clinical lentivirus production;
(3) the culture and culture method provided by the invention has the advantages that the serum-free medium is not required to be replaced in the culture process, the serum-free domestication of the working cells is not required, the culture and culture method is simple and convenient, and the consumed time is short.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
In order to better understand the concept and relationship of the subjects in the embodiments, the specific invention refers to: (1) the transfer vector carrying the gene of interest is co-transfected with a packaging vector into packaging cells (e.g., HEK293T cells in this example) and cultured in conventional medium (e.g., 10% FBS DMEM in this example). (2) The transfected packaging cells were cultured not in the conventional medium (e.g., FBS-containing DMEM medium in this example), but in 1% glutamine-supplemented VaccieXprees medium, and the virus was collected. (3) The virus transduces a target cell, such as a T lymphocyte or other host cell (e.g., lymphocytes (PBMCs) in this example 4). (4) The gene of interest is integrated and expressed in the target cells (e.g., lymphocytes (PBMC) as in example 4).
Example 1 HEK293T cell subculture
(1) Preparing a DMEM medium (10% DMEM for short) containing 10% FBS;
(2) reviving the HEK293T cells;
(3) the recovered HEK293T cells were added into 10ml 10% DMEM medium, centrifuged at 200-300g for 5 minutes, the supernatant was discarded, the cells were resuspended in 10% DMEM and transferred to a cell culture flask, carbon dioxide incubator, 37 ℃ C, 5% CO2Performing condition culture;
(4) when the cell density reaches a certain degree, cell passage is carried out to maintain the good state of the cells.
Example 2 Lentiviral preparation
(1) 1 x 10^6HEK293T cells/hole are planted in a 6-hole plate, when the cell fusion degree in the hole plate reaches 60-80%, original culture medium in the hole plate is removed, and the hole plate is replaced by DMEM culture medium containing 5% FBS (hereinafter, 5% DMEM) for about 2 hours;
(2) adding the packaged plasmid, the target plasmid and 2.5M CaCl into a 15ml centrifuge tube2Adding sterilized ultrapure water into the solution mixture, mixing uniformly, slowly adding a 2 x HBS solution with the pH value of 6.90-7.10 into the solution mixture, oscillating, mixing uniformly, slowly dripping, mixing uniformly, standing and incubating for 5-10 minutes to form a calcium phosphate-DNA compound;
(3) adding 200 μ l of calcium phosphate-DNA complex into each well of 6-well plate, mixing, placing in carbon dioxide incubator at 37 deg.C and 5% CO2Culturing for 3-8 hr;
(4) taking out 6-well plate, removing supernatant, adding 5% DMEM, 1% glutamine additive-containing vaccine Xprees culture medium, Gibco FreeStyle 293 culture medium, Gibco VP-SFM culture medium, OPTI PRO-SFM culture medium 2ml into different wells respectively;
(5) viral supernatants were harvested 40-48 hours after plasmid transfection.
Example 3 Lentiviral titer assay
(1) HEK293T cells were digested, resuspended and counted, and the number of plated cells per well was 1 × 105.
(2) After 3-6 hours, after the cells in the 24-well plate were attached, 10. mu.l of the lentiviral supernatant produced in example 2 was added to the well, and 1. mu.l of Polybrene was added thereto at 37 ℃ with 5% CO2Conditioned for 2 days.
(3) After 2 days, the HEK293T cells in the 24-well plate are digested, and the positive rate of the cells is detected by flow; the detection reagent is Protein-L.
Protein-L can specifically recognize an immunoglobulin light chain kappa chain, and a single-chain antibody sequence contained in the lentiviral vector can be recognized by the Protein-L; if 293T cells express the CAR structure in the viral vector, it will be detected by the marker. Pro-L positive rate indicates the virus infectivity, and the stronger the infectivity of the same virus vector to the same cell, the higher the virus titer at the same volume of virus supernatant. Viral infectivity the conversion equation before viral infectivity and titer was:
the unit of virus titer is TU/ml;
the volume of virus is in ml.
The results of the experimental tests are shown in table 1:
TABLE 1 infection efficiency of cultured viruses of different cultures
And (4) conclusion: the vaccine Xprees culture medium can be used for preparing lentivirus, and the infection capacity of the lentivirus can be obviously improved by using the culture medium, which is far higher than other serum-free culture media and conventional DMEM culture medium with 5% FBS. Thus the use of the vacceneXprees medium for lentivirus production enables higher virus yields than conventional production protocols.
Example 4 viral transduction of target cells
Culturing newly prepared mononuclear cell PBMC in 10% FBS-containing RPMI 1640 complete culture medium, and activating anti-CD 3 and anti-CD 28 monoclonal antibodies to perform lentivirus infection; separately adding the prepared lentivirus containing the target gene and uninfected peripheral blood lymphocytes (PBMC) as blank control; after 24h, the medium was replaced with RPMI 1640 complete medium containing 500IU/mL recombinant human IL-2, the culture was continued for 10-20 days, and the expression of the target gene was detected by Protein-L on the 10 th day of the culture.
Comparative example 1 VaccineXpress Medium and DMEM Medium Single use test
According to the method of example 2 and example 3, the combination of the VaccieXpress medium protocol was changed to two changes of the VaccieXpress medium and 5% DMEM 2 changes to perform the culture test and detection. The results of the detection of the cultured lentiviral vectors are shown in Table 2 below.
TABLE 2 transfection efficiency of viruses grown in Single Medium
| Experimental group | Vaccinexpress2 times of liquid change | 5% DMEM 2 exchange liquid |
| 1 | 9.70％ | 16.00％ |
| 2 | 9.40％ | 10.70％ |
And (4) conclusion: the ability to prepare virus using the vacceneXpress medium 293T cells was inferior to that of the conventional 5% DMEM medium, and the preparation scheme using DMEM + VacconeiXpress and the preparation scheme using DMEM alone were superior, so that the preparation scheme of DMEM + VacconeiXpress was optimal in combination with the scheme of example 3, and thus the present invention first uses a DMEM medium containing FBS to exchange the medium after adding the calcium phosphate-DNA complex and culturing for 3-8 hours, and then exchanges the VacconeiXpress medium to culture lentivirus.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (18)
1. A combination of media suitable for large scale clinical grade viral vector production, wherein said media comprises conventional media and vaccenexpress media.
2. The medium according to claim 1, wherein the conventional medium is a DMEM medium containing FBS.
3. The media combination of claim 1, wherein glutamine is added to the vacunexpress media.
4. The media combination of claim 1, wherein the vacceneXpress media is packaged individually.
5. A method for reducing the residual amount of bovine serum albumin in viral particles using the culture medium composition according to any one of claims 1 to 4, wherein a cell line producing viral particles is cultured in said VaccieXpress medium.
6. The method of claim 5, wherein the cell line is a HEK293T cell.
7. The method according to claim 5, characterized in that it comprises in particular the steps of:
(1) pretreatment of
Culturing a cell line which can generate virus particles and is to be transfected by the conventional culture medium, and preparing a packaging plasmid and a slow virus plasmid carrying a target gene into a calcium phosphate-DNA compound;
(2) transfected cells
Adding the calcium phosphate-DNA complex into the cell line which can generate the virus particles and is cultured by the conventional culture medium, and obtaining the cell line which can generate the virus particles after the cell line is transfected, wherein the cell line which can generate the virus particles comprises the slow virus vector of the target gene;
(3) collection of viruses
The cell line producing virus particles is cultured in the VaccireXpress medium, and the virus is collected.
8. A virus produced by the method of claim 5 and a host cell for viral use.
9. The host cell of claim 8, wherein the host cell is a T cell.
Use of a vacceneXpress medium as a medium for said cell line capable of producing viral particles.
11. The use according to claim 10, wherein glutamine is added to said vacuneexpress medium.
12. A composition suitable for large scale clinical grade viral vector production, said composition comprising said vaccenexpress medium and said viral particle producing cell line.
13. The composition of claim 12, wherein the cell line is a HEK293T cell.
14. The composition of claim 12, wherein said composition consists of said vacceneXpress medium, glutamine and said cell line that produces viral particles.
15. A method for improving the infection efficiency of a lentiviral vector, which is characterized in that a cell line capable of producing viral particles is transfected by the lentiviral vector containing a target gene under the conventional culture medium condition, and the cell line is cultured by the VaccieXpress culture medium, and the supernatant is the viral particles produced by the cell line.
16. The method of claim 15, wherein the gene of interest comprises a chimeric antigen receptor nucleic acid sequence or a gene therapy related nucleic acid sequence.
17. The method of claim 15, wherein said vacunexpress medium contains 1-5% by volume of said glutamine.
18. The method according to claim 15, characterized in that it comprises the steps of:
(1) culturing cells to be transfected by using a DMEM medium containing FBS; preparing the packaging plasmid and the lentivirus plasmid carrying the target gene into a calcium phosphate-DNA compound;
(2) adding the calcium phosphate-DNA compound into the cells to be transfected, and culturing the cells by using the DMEM medium containing FBS to obtain a cell line capable of producing virus particles, wherein the cell line capable of producing virus particles carries a lentiviral vector of a target gene;
(3) the virus particle-producing cell line was subjected to supernatant removal and continued to culture in glutamine-containing Vaccinenes medium.
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| CN114214287A (en) * | 2021-12-28 | 2022-03-22 | 广州泛恩生物科技有限公司 | Liquid exchange medium and application thereof |
| WO2023051607A1 (en) * | 2021-09-29 | 2023-04-06 | 上海行深生物科技有限公司 | Virus culture method |
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