阅读说明：本技术 人源细胞悬浮培养的培养基组合及溶瘤痘苗病毒的制备方法 (Culture medium composition for human-derived cell suspension culture and preparation method of oncolytic vaccinia virus ) 是由 曾宇明 范琦 李坤 刘冬连 于 2020-06-16 设计创作，主要内容包括：本发明涉及溶瘤病毒制备技术领域,尤其涉及人源细胞悬浮培养的培养基组合及溶瘤痘苗病毒的制备方法。本发明提供的培养基组合,包括EX-Cell<Sup>? </Sup>293培养基、VirusPro<Sup>?</Sup>CD HeLa培养基、DMEM培养基中至少两种。该培养基组合能够使悬浮培养的HeLa S3细胞能保持高密度高活率生长,并且支持搅拌式生物反应器大规模培养细胞扩增病毒。同时,悬浮培养HeLa S3细胞扩增痘苗病毒,病毒滴度也稳定在较高水平,实现了溶瘤痘苗病毒的大规模生产。(The invention relates to the technical field of oncolytic virus preparation, in particular to a culture medium composition for human cell suspension culture and a preparation method of oncolytic vaccinia virus. The culture medium combination provided by the invention comprises EX-Cell ® 293 Medium, VirusPro ® The combination of the culture media can ensure that He L a S3 cells cultured in suspension can keep high-density and high-activity growth, and support a stirring bioreactor to culture cells in large scale and amplify viruses, and meanwhile, the He L a S3 cells cultured in suspension amplify the vaccinia viruses, so that the virus titer is also stabilized at a higher level, and the large-scale production of the oncolytic vaccinia viruses is realized.)

1. Use of a combination of media consisting of EX-Cell in He L a Cell suspension culture^®293 Medium, VirusPro^®CD He L a culture medium and DMEM culture medium.

2. A combination of media for He L a cell suspension culture that is:

EX-Cell^®293 medium and DMEM medium; wherein EX-Cell^®The volume ratio of the 293 medium to the DMEM medium is 1:2, 1:1 or 2: 1;

or EX-Cell^®293 Medium and VirusPro^®CD He L a culture medium composition, wherein EX-Cell^®293 Medium and VirusPro^®The volume ratio of the CD He L a culture medium is 1:2, 1:1 or 2: 1;

or VirusPro^®Combination of CD He L a culture medium and DMEM culture medium, wherein VirusPro^®The volume ratio of the CD He L a culture medium to the DMEM culture medium is 1:2, 1:1 or 2: 1;

or EX-Cell^®293 Medium, VirusPro^®Combination of CD He L a medium and DMEM medium, wherein EX-Cell^®293 Medium, VirusPro^®The volume ratio of the CD He L a culture medium to the DMEM culture medium is 1:1:1, 1:2:1, 2:1:1 or 1:1: 2.

3. According to claim 2The culture medium combination described above, the EX-Cell^®293 Medium, VirusPro^®The CD He L a culture medium or DMEM culture medium is also added with factors for promoting cell growth;

the factor is selected from insulin, growth hormone, epidermal growth factor, fibroblast growth factor, proliferation stimulating factor, and insulin-like growth factor.

4. Use of a combination of media according to claim 2 or 3 for the preparation of an oncolytic vaccinia virus.

A method for suspension culture of He L a cells, which comprises inoculating suspension-acclimatized He L a cells into the culture medium combination according to claim 2 or 3, and culturing the cells with shaking.

6. A method for producing an oncolytic vaccinia virus, comprising inoculating a suspension acclimatized He L a cell to the culture medium composition of claim 2 or 3, and culturing the cell with shaking until the cell density is not less than 5 × 10⁶cells/ml, then dilute the cells to a density of 2 × 10⁶cells/ml ~5×10⁶Inoculating cells/ml with virus, and continuing culturing for 2-3 d to recover the virus.

7. The method of claim 6, wherein the cells are diluted to a density of 3 × 10⁶cells/ml ~4×10⁶cells/ml were inoculated with virus.

8. The preparation method according to claim 6, wherein the shaking culture conditions are 34-38 ℃ and 3-10% CO₂Culturing at 100-130 rpm for 24-96 hr, and subculturing or inoculating virus.

9. The method according to any one of claims 6 to 8, wherein the MOI of the inoculated virus is 0.001 to 0.5 pfu/cell.

10. The method of claim 6, wherein the virus is a vaccinia L ister strain or a genetically modified recombinant vaccinia L ister strain.

Technical Field

The invention relates to the technical field of oncolytic virus preparation, in particular to a culture medium composition for human cell suspension culture and a preparation method of oncolytic vaccinia virus.

Background

Oncolytic Virus (oncolytical Virus) can kill tumor cells in a targeted manner without killing normal tissue cells. Meanwhile, the oncolytic virus can kill the anti-apoptosis tumor cells, does not generate antagonism on the existing anti-tumor treatment scheme, and is suitable for combined medication.

Vaccinia Virus (vaccius Virus, VACV) has been widely used in the world for killing smallpox, and humans have a deep understanding of the biological properties and pathogenic mechanism of Vaccinia Virus, and meanwhile, Vaccinia Virus has fast replication, clear side effects, large self-genome, and can be inserted into large segments of exogenous genes, and is suitable for tumor treatment after being modified.

In recent years, some researchers at home and abroad have devoted to the application of recombinant vaccinia virus (rVACV) to the biological treatment of tumors, some of which have made major breakthroughs, and in particular, French Transgene company has made the research on oncolytic vaccinia virus on the list that although the oncolytic vaccinia virus item JX594 (Pexa-Vec) failed in the clinical stage III liver cancer, TG6002, a product of the same type, was undergoing clinical trials on colorectal cancer I/IIa in the United kingdom, the product used a new gene editing technique and removed two genes, and expected to have a better therapeutic effect.

At present, host cells for culturing oncolytic vaccinia virus comprise BSC-1, Vero, He L a S3, MRC-5, BHK-21 and the like, but human cells are few and mostly grow adherently, serum is almost required to be added into a culture medium, the complexity and uncertainty of serum components and the potential risk of pollution of pathogenic microorganisms such as virus exist in the culture medium, the production process and quality of the virus are influenced, and the use of the oncolytic virus brings non-negligible safety hazards.

With the continuous progress of the serum-free culture technology, a plurality of cells (such as CHO cells, HEK293 cells, BHK-21 cells and the like) can realize serum-free suspension culture, but the anchorage characteristics of a plurality of cells (such as VERO cells) are difficult to change, and the culture mode still adopts the inner surfaces of square bottles and roller bottles as media for adhesion growth. The cell growth area that these culture systems can provide is limited, and the enlargement of cell culture scale can only rely on the increase of culture unit, therefore bring to virus production such as the pollution risk is big, production efficiency is low, product quality controllability is poor and manpower resources demand is big etc. drawback. Microcarrier culture is a specialized cell adherent culture mode which combines the advantages of suspension culture, not only greatly increases the surface area on which cells grow in unit culture volume, but also increases the implementable degree of monitoring and controlling the cell culture process and amplifying the cell culture scale. However, compared with suspension culture, the use of microcarriers also increases the cost of cell culture, introduces a lot of uncontrollable risks, and also brings inconvenience to the scale-up of the culture.

In addition, He L a S3 cells are sensitive to shear force, can grow well by using shake flask culture during suspension culture, and cannot grow normally once being cultured in a reactor under stirring, and the cell viability is sharply reduced.

Disclosure of Invention

The invention aims to solve the technical problems of providing a culture medium combination for human cell suspension culture and a preparation method of oncolytic vaccinia virus, and the culture medium can be used for domesticating He L a cells growing adherently, so that the cells are adapted to serum-free suspension culture, the cells grow at high density and high survival rate, and the cells are cultured in a stirring bioreactor in a large scale to amplify the virus.

The invention provides application of a culture medium combination in He L a Cell suspension culture, wherein the culture medium combination is formed by EX-Cell^®293 Medium, VirusPro^®CD He L a culture medium and DMEM culture medium.

The culture medium combination provided by the invention comprises EX-Cell^®293 Medium, VirusPro^®At least two of CD He L a culture medium and DMEM culture medium^®293 medium and DMEM medium; or EX-Cell^®293 Medium and VirusPro^®Combinations of CD He L a medium, or VirusPro^®Combination of CD He L a medium and DMEM medium, or EX-Cell^®293 Medium, VirusPro^®CD He L a medium and DMEM medium.

EX-Cell in the invention^®293 Medium, VirusPro^®The CD He L a culture medium and the DMEM culture medium are serum-free culture media, and serum and similar components are not added in the process of culturing the He L a cells^®293 Medium, VirusPro^®The CD He L a culture medium or DMEM culture medium can also be added with factors for promoting cell growth, wherein the factors are selected from insulin, growth hormone, epidermal growth factor (such as rHuEGF), fibroblast growth factor, proliferation stimulating factor (MSA), insulin-like growth factor (such as rHuIGF) and the like, the concentration of the factors for promoting cell growth is considered according to specific conditions, such as growth conditions, product requirements of the factors and the like, such as the addition of rHuEGF with the concentration of 40ng/ml, but the concentration is not limited to the above concentration.

The volume ratio of each medium in the medium composition provided by the present invention is not limited, and the medium composition in the example was verified to be an equal ratio.

In some embodiments, EX-Cell^®293 media in combination with DMEM Medium, EX-Cell^®293 cultivationThe volume ratio of the medium to the DMEM medium was 1: 1.

In some embodiments, EX-Cell^®293 Medium and VirusPro^®In combination with CD He L a Medium, EX-Cell^®293 Medium and VirusPro^®The volume ratio of the CD He L a culture medium is 1: 1.

In some embodiments, VirusPro^®VirusPro in combination with CD He L a Medium and DMEM Medium^®The volume ratio of CDHe L a medium to DMEM medium is 1: 1.

In some embodiments, EX-Cell^®293 Medium, VirusPro^®EX-Cell in combination of CD He L a Medium and DMEM Medium^®293 Medium, VirusPro^®The volume ratio of the CD He L a culture medium to the DMEM culture medium is 1:1: 1.

The culture medium combination is applied to the suspension culture of He L a cells, and the He L a cells are He L a S3 cells.

The invention also provides a method for He L a cell suspension culture, which comprises the steps of inoculating the suspension domesticated He L a cells into the culture medium, and performing shaking culture.

The preparation method of suspension domesticated He L a Cell comprises resuscitating and culturing He L a Cell in DMEM containing serum, and adding EX-Cell^®293 heavy suspension cells for suspension acclimation.

In the invention, the conditions of the shaking culture are 34-38 ℃ and 3-10% CO₂Under the condition, shaking culture is carried out for 24-96 hours at 100-130 rpm. In the present example, the shaking culture was carried out at 37 ℃ under 5% CO₂Subcultured 3d (72 hours) at 110 rpm.

In the invention, the inoculation MOI is 0.001 pfu/cell-0.5 pfu/cell.

In some embodiments, the detoxification MOI is 0.005 pfu/cell to 0.1 pfu/cell. In some embodiments, the MOI of the inoculation is 0.02 pfu/cell.

The culture medium disclosed by the invention is applied to preparation of oncolytic vaccinia virus.

The invention also provides a preparation method of the oncolytic vaccinia virus, which comprises the following steps:inoculating suspension domesticated He L a cells into the culture medium, and performing shake culture until the cell density is not lower than 5 × 10⁶cells/ml, then dilute the cells to a density of 2 × 10⁶cells/ml ~5×10⁶Inoculating cells/ml with virus, and continuing culturing for 2-3 d to recover the virus.

According to the culture method of the He L a cells, provided by the invention, the high-density He L a cells are diluted to a proper density and then viruses are amplified, so that the culture volume of cell seeds is greatly reduced, the operation is simplified, and the labor intensity is reduced.

Culturing cells by suspension culture of He L a S3 cells to a density of not less than 5 × 10⁶cells/ml, e.g. 6 × 10⁶cells/ml~7×10⁶cells/ml, diluted to a density of 2 × 10⁶cells/ml ~5×10⁶cell/ml inoculation;

in some embodiments, the dilution is by a combination of media used in culture, and in some embodiments, the density of He L a S3 cells at the time of inoculation is 2 × 10⁶cells/ml ~5×10⁶cells/ml、3×10⁶cells/ml ~4×10⁶cells/ml or 4 × 10⁶cells/ml ~5×10⁶cells/ml. preferably, the cell density at the time of inoculation is 3 × 10⁶cells/ml ~4×10⁶cells/ml, more preferably, the density of cells at the time of inoculation is 3 × 10⁶cells/ml or about 4 × 10⁶cells/ml。

Virus inoculation: the virus amplification method comprises the following steps: the MOI is: 0.001 pfu/cell-0.5 pfu/cell, temperature: CO at 32-38 DEG C₂3-10%, and the culture time is as follows: 1-3 days. Preferably, the MOI is 0.01-0.1 pfu/cell, the temperature: 37 ℃ CO₂: 5%, and culturing for 2 days.

And (3) harvesting the virus: centrifuging, removing supernatant, harvesting cells, breaking cells, centrifuging or filtering to remove cell debris, and harvesting virus liquid.

In addition, the method provided by the invention is also suitable for large-scale culture, so that the vaccinia virus can be produced in a large scale, the single-cell virus production capacity is equivalent to that of shake flask virus expansion, and a higher level is kept. The production method comprises the following steps:

(1) culture of cells suspension culture of He L a S3 cellsDensity of 5 × 10⁶cells/ml ~7×10⁶cells/ml, then dilute the cells to a density of 3 × 10⁶cells/ml ~4×10⁶cells/ml can be inoculated with virus.

(2) Virus inoculation: the virus amplification method comprises the following steps: the MOI is: 0.001 pfu/cell-0.5 pfu/cell, temperature: 32-38 ℃, DO: 30-80%, pH 6.8-7.6, and culture time is as follows: 1-3 days. Preferably, the MOI is 0.01-0.1 pfu/cell, the temperature: 37 ℃, DO: 50%, pH 7.4, cultured for 2 days.

(3) And (3) harvesting the virus: centrifuging, removing supernatant, harvesting cells, breaking cells, centrifuging or filtering to remove cell debris, and harvesting virus liquid.

The vaccinating virus is a vaccinia virus, either modified or unmodified, and in the present invention, the virus is a vaccinia L ister strain.

The culture medium combination provided by the invention comprises EX-Cell^®293 Medium, VirusPro^®The combination of the culture media can ensure that He L a S3 cells cultured in suspension can keep high-density and high-activity growth, and support a stirring bioreactor to culture cells in large scale to amplify viruses, and meanwhile, the He L a S3 cells cultured in suspension can amplify the vaccinia viruses, so that the virus titer is also stabilized at a higher level, and the large-scale production of the oncolytic vaccinia viruses is realized.

Drawings

FIG. 1He L a S3 cell growth curves.

Detailed Description

The invention provides a culture medium combination for human cell suspension culture and a preparation method of oncolytic vaccinia virus, and a person skilled in the art can use the contents for reference and appropriately improve process parameters to realize the culture. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market. Wherein, EX-Cell^®293 Medium from Sigma, DMEM Medium from Sigma, VirusPro^®CD He L a was purchased from Yuanbei Biotech, Inc.

The invention is further illustrated by the following examples: