CN115466710A

CN115466710A - Method for obtaining virus-free cell line and virus-free cell line obtained by method

Info

Publication number: CN115466710A
Application number: CN202210650493.8A
Authority: CN
Inventors: 杨慧; 李莉莉
Original assignee: Beijing Solobio Genetechnology Co Ltd; Staidson Beijing Biopharmaceutical Co Ltd
Current assignee: Beijing Solobio Genetechnology Co Ltd
Priority date: 2021-06-11
Filing date: 2022-06-09
Publication date: 2022-12-13

Abstract

The invention discloses a method for establishing a virus-free cell line from a virus-contaminated organism or cell population, and also provides a virus-free cell line which is established according to the method and is derived from the virus-contaminated organism or cell population. An exemplary method for establishing a virus-free cell line comprises obtaining cells from a virus-contaminated organism or cell population, washing and resuspending the cells, separating the cells into single cells or multiple cells, culturing and expanding the single cells or multiple cells obtained by separation in a cell culture medium containing an amantadine compound to form single-cell clones or multiple-cell clones, detecting whether viruses exist in the cell clones or the cell culture medium, inoculating the cell clones detected to have no viruses in the cell culture medium containing no amantadine compound, and continuing the culture and expansion to obtain the virus-free cell line.

Description

Method for obtaining virus-free cell line and virus-free cell line obtained by method

Technical Field

The present invention relates to a method for obtaining a virus-free cell line derived from an organism or a cell population contaminated with a virus, and to a virus-free cell line obtained using said method.

Background

Insect cells are widely used for expression of recombinant proteins, typically as hosts for recombinant baculovirus vectors, and also for plasmid-mediated transient transfection or stable genetic transformation. Insect cells are useful for the scientific expression of proteins, and for the manufacture of human and veterinary biologics. Baculovirus-insect expression system (BICS) is a kind of eukaryotic expression system with wide application, which is an expression system taking baculovirus as exogenous gene vector and insect cell as receptor, has the characteristics of safety, high efficiency, large capacity, high expression level, capability of folding and modifying expression products and the like, and is widely applied in the field of biomedicine. Recently, several insect cell lines for recombinant protein expression were discovered to be persistently infected with foreign viruses, raising questions about how these infections might affect studies conducted using these cell lines. In addition, these findings raise a great deal of attention to the safety of biologicals produced using these cell lines.

Sf-Rhabdovirus (Sf-Rhabvirus) was independently found in different Sf (Spodoptera frugiperda) cell lines by three different teams at the same time. Sf-rhabdovirus is a typical rhabdovirus with a single (-) strand genome of approximately 13.5kb in size, encoding in sequence a typical rhabdovirus nucleoprotein (N), phosphoprotein (P), matrix (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). Between G and L, there is an additional open reading frame, designated "X" and encoding a putative viral sporosin, which is likely to be lost after serial passage of infected Sf cells, whereas it is not required for in vitro infection of Sf-rhabdovirus.

New insect cell lines free of exogenous viruses have been isolated for use as improved research tools and safer bio-manufacturing platforms. To completely remove Sf-rhabdovirus from Sf9 (Spodoptera frugiperda 9) cells, grilicobeck company treated the starting culture consisting of several cells with nucleoside drugs and further expanded culture yielded Sf-rhabdovirus-free cell lines with the drug 6-azauridine finally used (patent application No. CN 201680071306.3). However, the patent application tries a method of co-inhibiting by using a plurality of drugs in actual screening, and the application of a plurality of antiviral drugs influences the cell metabolism and biosynthesis process, aggravates cell damage and influences the capacity of cell recombination and virus expression after screening. Although other studies have obtained a cell line with similar growth characteristics and better viability by screening without the addition of antiviral drugs (patent application No. CN 201910317758.0), the efficiency of obtaining a virus-free cell line by this method is very low.

Therefore, there is still a need in the art for methods to efficiently and stably obtain and establish virus-free cell lines from virus-contaminated organisms or cell populations, and virus-free cell lines that retain their original cellular biological properties and functions, such as cell viability and ability to express foreign recombinant proteins or produce recombinant viruses.

Disclosure of Invention

The invention is based on the following findings of the inventors: when establishing virus-free cell lines from virus-contaminated organisms or cell populations, the type of antiviral drug used and its concentration, and the number of cells in the initial culture are critical to the efficiency of establishing virus-free cell lines and whether the established cell lines retain their cellular biological properties and functions.

Thus, the present invention provides a method for efficiently and stably establishing a virus-free cell line from a virus-contaminated organism or cell population, wherein the cell line is devoid of viruses while maintaining relevant cellular biological properties and functions (e.g., cell viability, ability to produce recombinant viruses or express recombinant proteins, etc.), and cell lines established using the method.

In one aspect, the invention provides a method for obtaining and establishing a virus-free cell line from a virus-contaminated organism or cell population, comprising the steps of: obtaining cells from organisms or cell populations polluted by viruses, washing and resuspending the cells, separating the cells into single cells or multiple cells, culturing the single cells or multiple cells in a cell culture medium containing a certain concentration of amantadine compounds for a period of time to form single cell clones or multiple cell clones, taking out partial cells and a supernatant culture medium from the cell clones, detecting whether the viruses exist or not, inoculating the cell clones detected that the viruses do not exist in the culture medium containing no amantadine compounds, and continuously culturing and amplifying to obtain a virus-free cell line.

In another aspect, the virus contaminating the organism or cell population described in the present invention is an Sf-rhabdovirus. In another aspect, the virally-contaminated organisms of the invention are insects, preferably Spodoptera frugiperda (Sf); the virus-contaminated cell population is an established cell line or cell strain, such as a commercially available or marketed cell line, preferably an insect cell line, more preferably an Sf cell line (e.g., sf9 cell line, sf21 cell line, etc.).

Thus, the cell line of the invention may be derived directly or indirectly from Spodoptera frugiperda, or may be derived from a commercially available or commercial Sf9 cell line (which in the context of the invention may be denoted SF-Rha, meaning an Sf9 cell line containing Sf-rhabdovirus).

Further, the present invention provides in another aspect a virus-free Sf cell line (referred to herein as SF-RVN cell line) derived from a commercially available or commercial Sf9 cell line and established by the above method. Compared with the original Sf9 cell line containing Sf-rhabdovirus, the cell line established by the invention is lack of Sf-rhabdovirus, has the same or basically the same cell viability, cell diameter and cell agglomeration rate as the original Sf9 cell, and has the same or basically the same or even better baculovirus passage stability and recombinant AAV yield as the Sf9 cell.

The invention is further described with reference to the following drawings and detailed description, but is not limited thereto. All technical equivalents which may be substituted for elements thereof according to the disclosure are intended to be encompassed by the present patent.

Drawings

FIG. 1 is a comparison of cell viability in which SF-RVN indicates Sf9 cells selected without Sf-rhabdovirus and SF-Rha indicates Sf9 cells contaminated with Sf-rhabdovirus.

FIG. 2 is a comparison of cell diameters, in which SF-RVN indicates Sf9 cells selected without Sf-rhabdovirus and SF-Rha indicates Sf9 cells contaminated with Sf-rhabdovirus.

FIG. 3 is a comparison of cell clumping rates, where SF-RVN indicates Sf9 cells selected without Sf-rhabdovirus and SF-Rha indicates Sf9 cells contaminated with Sf-rhabdovirus.

FIG. 4 is a comparison of the passage stability (ratio of target genome titer to baculovirus genome titer) of baculoviruses, where SF-RVN indicates Sf9 cells selected without Sf-rhabdovirus and SF-Rha indicates Sf9 cells contaminated with Sf-rhabdovirus.

FIG. 5 shows a comparison of the yields of recombinant AAV, wherein SF-RVN indicates Sf9 cells selected without Sf-rhabdovirus and SF-Rha indicates Sf9 cells contaminated with Sf-rhabdovirus.

Detailed Description

To facilitate understanding of various embodiments of the inventive concepts, specific terms have the meanings set forth below.

Cell line: refers to a cell population expanded from one or several common ancestor cells, including but not limited to a cell population expanded from a single isolated cell.

Established cell lines: refers to a cell line that has the potential to proliferate indefinitely when cultured under appropriate conditions. Such cell lines have undergone changes (e.g., transformation, etc.) in vitro as compared to cells naturally occurring in an organism. The second cell line obtained by isolating a single cell from the first cell line and then expanding the isolated cell is sometimes referred to as a subclone of the first cell line.

Derived from an organism: means obtained directly or indirectly from an organism. The cells may be derived directly from the organism, e.g. by obtaining a tissue or organ from the organism and then lysing the tissue or organ to obtain primary cells. The cells may also be indirectly derived from the organism, for example, by isolating single cells from a cell line derived from the organism and then expanding the isolated single cells to establish and obtain a cell line.

The organism of the invention may be an insect, in particular a lepidopteran insect. Lepidopteran insects refer to any member of the order lepidopteran, including butterflies, moths, etc., whose adults have four broad or lanceolated wings, typically covered with tiny overlapping and brightly colored scales, and whose larvae are caterpillars. Lepidopteran insects include, but are not limited to, spodoptera frugiperda, trichoplusia ni, or Bombyx mori.

Single cell clones are defined as cell populations that are formed by the expansion of individual cell cultures. Polyclonal cells refer to a population of cells formed by the expansion of a plurality of cells in culture.

"comprising" or "including" is synonymous and is an open-ended term that does not exclude the presence of other unrecited components, elements, steps, or the like. For example, a composition "comprising" components a, B and C may consist of components a, B and C; alternatively, the composition may comprise not only components a, B and C, but also one or more other components.

The term "substantially the same" or "substantially equivalent" refers to a change in the ten percent range or no statistically significant difference relative to a particular condition or parameter. For example, but not by way of limitation, when a cell line obtained by the method of the present invention is propagated under the same conditions as the original cell or cell line (e.g., sf9 cell line) and the cell viability is determined by the same method, the viability of the established cell line is in the range of between 90% and 110% of the viability of the original cell, or there is no statistical difference between the two, based on statistical analysis; or when the established cell line and the original cell or cell line are propagated under the same conditions and the mean cell diameter is determined in the same way, the mean diameter of the established cell line is in the range between 90% and 110% of the mean diameter of the original cell, or there is no statistical difference between the two, based on statistical analysis.

The terms "detecting the presence of a virus", "detecting the presence of Sf-rhabdovirus" and related terms are used broadly in this specification. It will be appreciated by those skilled in the art that there are many detection techniques known in the art that can be used with the present invention. Exemplary techniques for detecting viruses include Polymerase Chain Reaction (PCR), reverse Transcription (RT), reverse transcription-polymerase chain reaction (RT-PCR), RT-PCR in combination with nested PCR, quantitative PCR (Q-PCR), RT-PCR in combination with quantitative PCR (quantitative RT-PCR or RT-QPCR), a variety of probe hybridization techniques, electron microscopy, and a variety of antibody-based detection techniques known in the art, such as ELISA assays. Detection techniques also include, but are not limited to, plaque assays and observation of cytopathic effect (CPE), bioinformatics techniques such as BLAST searches, and the like.

The amantadine compound refers to amantadine and derivatives thereof, and related salt forms, including but not limited to amantadine hydrochloride, rimantadine hydrochloride, amantadine or rimantadine.

In an exemplary embodiment of the invention, a method of obtaining a cell line lacking a virus comprises: obtaining cells from organisms or cell populations (including but not limited to cell lines or cell strains) polluted by viruses, washing and resuspending the cells, separating the cells into single cells or multiple cells, culturing the single cells or multiple cells in a cell culture medium containing a certain concentration of amantadine compounds for a period of time to form single cell clones or multiple cell clones, taking out partial cells or culture medium from the cell clones to detect whether the viruses exist, inoculating the cell clones detected that the viruses do not exist into the culture medium without the amantadine compounds to continue culture and amplification, and finally obtaining the cell lines without virus pollution.

In certain embodiments, the virus contaminating an organism or cell population described herein is an Sf-rhabdovirus. In some embodiments, the virally-contaminated organisms described herein are insects, preferably spodoptera frugiperda; the virus-contaminated cell population is an established cell line or cell strain, e.g. a commercially available or marketed cell line, preferably an insect cell line, more preferably an Sf9 or Sf21 cell line.

In certain embodiments, a single cell refers to only 1 cell; by plurality of cells is meant a cell containing several cells, including, but not limited to, 2-20 cells, preferably 3-10 cells, preferably 4-6 cells, more preferably 5 cells.

In certain embodiments, methods of isolating into a single cell or multiple cells include, but are not limited to, limiting dilution cloning (serial dilution cloning), cloning cells in soft agar and then picking cell colonies, cell sorting, laser Capture Microdissection (LCM), manual capture using a micropipette, microfluidics, or use of micromanipulators.

In certain embodiments, the isolated cell or cells are cultured in a cell culture medium of an amantadine-like compound, wherein the amantadine-like compound is amantadine hydrochloride. In certain embodiments the adamantanamine compound is adamantaneamine hydrochloride. In certain embodiments the adamantanamine-based compound is amantadine. In certain embodiments the adamantanamine-based compound is rimantadine.

In certain embodiments, the concentration of the amantadine compound in the cell culture medium containing the amantadine compound is 10 μ g/ml,20 μ g/ml,50 μ g/ml or 100 μ g/ml, preferably 50 μ g/ml.

In certain embodiments, methods for detecting the presence of a virus (including but not limited to Sf-rhabdovirus) are well known in the art and include, but are not limited to, RT-PCR, nested PCR, RT-PCR in combination with nested PCR, or RT-QPCR.

In some embodiments, after a period of time of the amplification culture of the cell clones tested for the absence of the virus in the medium without the adamantanamine compound, a portion of the cells of the amplification culture and the supernatant culture medium are again used to verify the complete removal of the virus.

In certain embodiments, the established virus-free cell lines are derived from primary cells contaminated with viruses, including but not limited to Sf-rhabdoviruses. In certain embodiments, the virus-contaminated primary cells are from a virus-contaminated organism (including, but not limited to, spodoptera frugiperda).

In other embodiments, the established virus-free cell lines are derived from a population of cells (including but not limited to commercially available or commercial cell lines) contaminated with viruses (including but not limited to Sf-rhabdoviruses). In certain embodiments, the virus-contaminated cell population is an Sf cell line contaminated with Sf-rhabdovirus, including but not limited to Sf9 or Sf21 cell lines.

In certain embodiments, the virus-free cell line established according to the methods of the invention (referred to herein as the SF-RVN cell line) has the following advantages: when cultured and incubated under the same conditions (as described in the examples herein), the virus-free cell line has the same or substantially the same cell viability rate, average cell diameter, cell clumping rate as the original cell or population of cells contaminated with virus. In certain embodiments, the virus-free cell lines and virus-contaminated primary cells or cell populations established according to the methods of the invention have substantially equivalent or superior results when compared to baculovirus passage stability assays and recombinant adeno-associated virus (AAV) production under identical conditions (as described in the examples herein).

Those skilled in the art will appreciate that materials and conditions suitable for growth and incubation of a particular cell type are known in the art, as are sources of acquisition. Such sources include, but are not limited to, cell culture manuals, commercial cell banks, or media suppliers. Suitable cell culture conditions can also be readily determined using methods known in the art.

The following examples are illustrative only and are not intended to limit the invention.

The techniques used in the following examples are, unless otherwise specified, conventional techniques known to those skilled in the art; the instruments, reagents, etc. used, unless otherwise specifically noted in this specification, are publicly available to those skilled in the art.

Example 1 recovery and culture of Virus-contaminated Primary insect cell lines

A frozen Sf9 cell (Life Technologies, inc.) is taken and rapidly thawed in a water bath at 37 ℃, 15ml of ESF AF culture medium (Expression systems, 99-300-01) is added into a 125ml shaking flask, the cell in the freezing tube is transferred into the shaking flask, the shaking flask is gently shaken and uniformly mixed, and the shaking flask is placed on a shaking table at 27-29 ℃ and 110-150 rpm for culture. Conventional culture was then maintained in ESF AF medium.

EXAMPLE 2 method for obtaining Sf cell lines deficient in Sf-rhabdovirus (SF-RVN)

1. Cell dilution:

the Sf9 cells cultured in example 1 were counted as in table 1:

TABLE 1

And (4) centrifuging the cell suspension, cleaning the cells, resuspending the cells by using a culture medium, counting the cells, and recording the cell density and the cell survival rate. The resuspended cells were taken and diluted 20-fold by adding medium.

2. Culture inhibition by amantadine hydrochloride or amantadine hydrochloride

The cells were subjected to virus-suppressed culture of a single cell or a plurality of cells (5 cells/well, 10 cells/well) isolated by limiting dilution with amantadine hydrochloride or rimantadine hydrochloride at the following concentrations: final concentration 1 cell/well, 5 cells/well, 10 cells/well; viral inhibitors: amantadine hydrochloride (final concentration 10. Mu.g/ml, 20. Mu.g/ml, 50. Mu.g/ml) (sigma, A1260-5G) or rimantadine hydrochloride (final concentration 10. Mu.g/ml, 20. Mu.g/ml, 50. Mu.g/ml) (sigma, 390593-1G).

The single cell or multiple cells are treated with amantadine hydrochloride or amantadine hydrochloride with different concentrations for about one month, during which liquid exchange and cell amplification are carried out to form single cell clone or multiple cell clone respectively, and then sampling is carried out to carry out Sf-rhabdovirus detection, wherein the detection method is as described in example 3.

As shown in Table 2, sf-free rhabdovirus cell lines (total 4 lines, 2 lines of amantadine hydrochloride and 2 lines of amantadine hydrochloride) were screened for each of 5 cells/well at a final concentration of amantadine hydrochloride or amantadine hydrochloride of 50. Mu.g/ml. The 4 selected strains of cells were transferred to a culture medium lacking antiviral compounds, named SF-RVN P0 generation cells when expanded to T25 square flask, and the adherent cells were subjected to suspension acclimation culture at P2 generation, while being subjected to serum-deprived acclimation culture, and then maintained to culture in SF900 ii SFM medium.

TABLE 2

Example 3 Sf-Rhabdoviral assay

To verify the presence of Sf-rhabdovirus in Sf-RVN cell lines, RNA was extracted from different passage levels of SF-RVN cells and supernatant and Sf-rhabdovirus was tested by RT-PCR (see: ma et al, J.Virol.88:6576-85, 2014).

In addition, in order to detect the presence of Sf-rhabdovirus more accurately and precisely, a set of primers and probes (Primer Premier 6.25) was designed based on the Sf-rhabdovirus L sequence (Genbank: KF 947078.1), and the one-step quantitative RT-PCR (RT-QPCR) method was used to quantitatively detect the Sf-rhabdovirus content. Firstly, respectively extracting RNA in cells and culture supernatant, and then adopting a TaqMan RNA-to CT 1-Step kit (ABI/Thermo, 4392938) to detect the content of the Sf-rhabdovirus by a one-Step method RT-QPCR according to the operation instruction of the kit.

Example 4 SF-RVN cell line deficient in Sf-rhabdovirus

RNA (including 2 cells selected from amantadine hydrochloride and 2 cells selected from amantadine hydrochloride) was extracted from SF-RVN cells and supernatants at different passage levels, respectively, and the presence of Sf-rhabdovirus was determined by the above method. When detected by RT-PCR, no Sf-rhabdovirus was detected in the selected Sf-RVNs (results not shown).

The results of RT-QPCR detection are shown in Table 3. From the above results, it was found that SF-RVN cells selected from amantadine hydrochloride or rimantadine hydrochloride were subjected to continuous subculture for 30 passages in a medium lacking in amantadine hydrochloride or rimantadine hydrochloride, RNA was isolated from a part of SF-RVN cells and supernatant every 5 passages, and Sf-rhabdovirus detection was performed, and Sf-rhabdovirus was not detected in any of 4 strains of cells, while Sf-rhabdovirus was detected in original Sf9 cells as a positive control.

The above results indicate that SF-RVN cells were subcultured for 30 passages in amantadine hydrochloride-deficient or amantadine hydrochloride-deficient medium, and no SF-rhabdovirus was detected in both cells and supernatant medium, indicating that SF-RVN cells are SF-rhabdovirus-free and that SF-rhabdovirus does not appear with cell passage and expansion.

TABLE 3

Example 5 Mycoplasma detection

The PCR method is adopted to detect the Mycoplasma in SF-RVN cells and culture supernatant, the Mycoplasma detection Kit is EZ-PCR-Mycoplasma-Test-Kit (Biological Industries, 20-700-20), and the specific method steps refer to the Kit instruction. The results showed that no mycoplasma contamination was detected in the selected SF-RVN cells.

Further detection and validation were carried out using 1 of the 4 SF-RVN cells described above (obtained via amantadine hydrochloride screening) as an example, as shown in examples 6-8. Similar tests and validations were performed on the other 3 cell lines, with results similar or equivalent to those of the cell line (obtained via amantadine hydrochloride screening) (results not shown).

Example 6 cell growth status, morphology and cell diameter

SF-RVN cells or SF-Rha cells (representing Sf9 cells containing Sf-rhabdovirus) were fixed at an initial density of 1.5X 10 per passage ⁶ cells/ml, volume 80ml,110rpm-150rpm,27 ℃ -29 ℃. At each passage, cells were examinedViability, cell diameter, and cell clumping rate.

And (3) displaying a detection result: there was no significant difference between SF-RVN cells and SF-Rha cells (representing Sf9 cells containing Sf-rhabdovirus) in cell viability rate (FIG. 1), cell diameter (FIG. 2), and cell clumping rate (FIG. 3) during cell culture. These results indicate that the selected SF-RVN cells are consistent with Sf9 cells in terms of essential cell growth characteristics.

Example 7 baculovirus expansion passage stability

Adjusting SF-RVN cells and SF-Rha cells (representing SF9 cells containing SF-rhabdovirus) to a certain density, adding a proper amount of cells into a six-well plate, culturing the cells at 27-30 ℃ until the cells adhere to the wall, then adding bacmid carrying EGFP exogenous genes for cell transfection, and harvesting P0-generation baculovirus virus seeds after certain transfection time. And (3) amplifying the P0 generation virus seeds to P1, P2, P3 and P4 generations according to a certain proportion, and detecting the genome titer of a target sequence (EGFP) and the genome titer of the baculovirus of each generation virus seed.

The specific method comprises the following steps: designing a specific primer probe aiming at a target sequence EGFP, and detecting the genome titer of the target sequence EGFP by a Q-PCR technology, and in addition, designing the primer probe aiming at a conserved sequence of the baculovirus and detecting the genome titer of the baculovirus by the Q-PCR technology. The ratio of the sequence of interest (EGFP) genomic titer to the baculovirus genomic titer was then calculated and compared (higher ratios indicate more stable passage).

As shown in FIG. 4, the screened SF-RVN cells have substantially equivalent to SF-Rha cells, and even better baculovirus passage stability.

Example 8 recombinant AAV viral production

Culturing SF-RVN cells and SF-Rha cells (representing SF9 cells containing SF-rhabdovirus) to a certain cell density, respectively adding a certain volume of baculovirus virus seeds (carrying AAV-Rep/AAV-Cap/EGFP exogenous genes), culturing at 110-150rpm and 27-29 ℃ for 72-120 h, and harvesting AAV. The genome titer of AAV was determined and compared.

The specific method comprises the following steps: and (3) taking a proper amount of purified AAV samples, inactivating DNase I, diluting by proper times, and then performing Q-PCR reaction, wherein the used primer is a specific primer probe aiming at EGFP.

As shown in FIG. 5, the selected SF-RVN cells are substantially equivalent to SF-Rha cells in terms of recombinant AAV yield, and are even more excellent. It can be seen that the ability of Sf-rhabdovirus-depleted SF-RVN cells obtained by screening to produce recombinant AAV is not affected at all.

From the results, it can be seen that Sf-rhabdovirus-free cell lines (SF-RVN cells) were obtained by screening with amantadine hydrochloride or rimantadine hydrochloride, which was concluded based on the results of highly sensitive RT-PCR and RT-QPCR assays, using the above method, that SF-RVN cells and supernatant medium did not have Sf-rhabdovirus detectable during at least 30 subcultures.

Functional studies are carried out on the screened SF-RVN cells, and the SF-RVN cells and the original Sf9 cells are found to be consistent in basic cell growth characteristics such as cell survival rate, cell diameter, cell agglomeration rate and the like, and are not polluted by mycoplasma. The SF-RVN cell is basically equivalent to the original Sf9 cell in the aspects of baculovirus passage stability, recombinant AAV virus yield and the like, and is even more excellent, which indicates that the activity of the Sf9 cell is not influenced by the method.

The results show that the screened SF-RVN cells can completely replace Sf9 cells and become safe production cells of proteins, viruses and vaccines.

Claims

1. A method of establishing a virus-free cell line from an organism or cell population derived from a viral contamination comprising:

obtaining cells from a virus-contaminated organism or cell population, washing and resuspending and isolating into a single cell or a plurality of cells;

culturing and amplifying the separated single cell or multiple cells in a cell culture medium containing the amantadine compound, and forming single cell clone or multiple cell clone;

removing a portion of said cell clone or said cell culture medium to detect the presence of a virus;

and inoculating the cell clone which does not exist in the virus to a cell culture medium which does not contain the amantadine compound for continuous culture and amplification, thereby obtaining a virus-free cell line.

2. The method of claim 1, wherein the virus comprises an Sf-rhabdovirus.

3. The method of claim 1 or 2, wherein the organism is an insect and the population of cells is an insect cell line.

4. The method of claim 3, wherein the insect comprises a lepidopteran insect.

5. The method of claim 4, wherein the lepidopteran insect comprises a Spodoptera frugiperda, a Trichoplusia ni, or a Bombyx mori silkworm.

6. The method of claim 1, wherein the cell line is derived from virus-contaminated primary cells.

7. The method of claim 1, wherein the cell line is derived from a virally-contaminated Sf cell line.

8. The method of any one of claims 1-7, wherein the virus-contaminated Sf cell line comprises Sf21 cells or Sf9 cells, and wherein the virus comprises Sf-rhabdovirus.

9. The method of any one of claims 1-8, wherein the detection method comprises: (a) RT-PCR; (b) RT-PCR and nested PCR; (c) quantitative RT-PCR; or (d) antibody-based detection techniques.

10. The method of any one of claims 1-9, wherein the amantadine-based compound is selected from at least one of: amantadine hydrochloride, rimantadine hydrochloride, amantadine, rimantadine.

11. The method of any one of claims 1-10, wherein the amantadine compound is amantadine hydrochloride or rimantadine hydrochloride.

12. The method of any one of claims 1-11, wherein the method of isolating into a single cell or a plurality of cells comprises: limiting dilution cloning, cloning cells in soft agar and then picking cell colonies, cell sorting, laser Capture Microdissection (LCM), manual capture using a micropipette, microfluidics or using a micromanipulator, preferably limiting dilution cloning.

13. The method according to claim 12, wherein the plurality of cells is 2-20 cells, preferably 3-10 cells, preferably 4-6 cells, more preferably 5 cells.

14. The method of any one of claims 1-13: wherein the concentration of amantadine hydrochloride or amantadine ethylamine hydrochloride is selected from 10 μ g/ml,20 μ g/ml or 50 μ g/ml.

15. The method of claim 13 or 14: wherein the plurality of cells is 5 cells; the concentration of amantadine hydrochloride or amantadine hydrochloride was 50. Mu.g/ml.

16. A virus-free cell line derived from a virus-contaminated organism or cell population, the cell line obtained by the method of any one of claims 1-15.

17. The cell line of claim 16, wherein the cell line is derived from an insect contaminated with a virus.

18. The cell line of claim 17, wherein the cell line is derived from a lepidopteran insect.

19. The cell line of claim 18, wherein the lepidopteran insect comprises a spodoptera frugiperda, a cabbage looper, or a bombyx mori.

20. The cell line of claim 19, wherein the cell line is derived from a spodoptera frugiperda cell line.

21. The cell line of claim 20, which is deficient in Sf-rhabdovirus.

22. The cell line of claim 21, further characterized by having the same or substantially the same cell viability rate, mean cell diameter, cell clumping rate as the virus-contaminated blasts or cell populations when propagated under the same conditions as the virus-contaminated blasts or cell populations.

23. The cell line of claim 21, further characterized in that the cell line has the same or substantially the same, or even better, virus passaging stability than the original cell or population of cells contaminated with virus when baculovirus infection is performed under the same conditions.

24. The cell line of claim 21, further characterized in that the cell line has the same or substantially the same, or even better, recombinant AAV production as compared to a virus-contaminated original cell or population of cells when recombinant AAV packaging is performed under the same conditions.

25. Use of a virus-free cell line as claimed in any one of claims 16 to 24 in a baculovirus-insect expression system.

26. Use of a virus-free cell line as claimed in any one of claims 16 to 24 for the expression of foreign recombinant proteins or for the production of recombinant viruses.