KR101831284B1 - Vero Cell Lines Suspension-cultivated Without Serum and Methods for Preparing Vaccine Virus With Those Cell Lines - Google Patents
Vero Cell Lines Suspension-cultivated Without Serum and Methods for Preparing Vaccine Virus With Those Cell Lines Download PDFInfo
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Abstract
본 발명은 ATCC CCL-81 세포주로서 기탁된 Vero 세포로부터 유도되고, 혈청 없이 배양이 가능하며, 부유 배양이 가능한 Vero 세포주, 세포주 Vero Sky7458 에 관한 것이다. 또한, 본 발명은 상기 Vero 세포를 성장시키는 배양 방법 및 상기 Vero 세포를 이용하여 백신용 바이러스를 제조하는 방법에 관한 것이다.The present invention relates to a Vero cell line, cell line Vero Sky7458, which is derived from Vero cells deposited as ATCC CCL-81 cell lines, is capable of culturing without serum, and is capable of suspension culture. The present invention also relates to a culture method for growing the Vero cells and a method for producing a vaccine virus using the Vero cells.
Description
본 발명은 무혈청 배지에서 부유 배양이 가능한 Vero 신규 세포주, 이러한 세포주를 제조하는 방법, 및 이러한 세포주를 이용하여 백신용 바이러스를 제조하는 방법에 관한 것이다.The present invention relates to a novel Vero cell line capable of suspension culture in a serum-free medium, a method for producing such a cell line, and a method for producing a vaccine virus using such a cell line.
일반적으로 백신의 생산을 위해서는 유정란, 마우스 뇌, 일차 세포 혹은 확립 세포가 사용된다. 이러한 전통적인 방법들은 몇 가지 문제를 가지고 있다. 예를 들어, 유정란을 사용할 경우 닭의 사육, 백신 생산 계획에 따른 수정란의 관리, 계란 단백질 유래 성분 제거를 위한 정제의 어려움 등의 문제가 있다. Generally, fertilized eggs, mouse brain, primary cells or established cells are used for vaccine production. These traditional methods have some problems. For example, when fertilized eggs are used, there are problems such as breeding of chickens, management of embryos according to vaccine production plan, and difficulty of refining to remove ingredients derived from egg protein.
세포 배양의 경우 일반적으로 증식 인자로 소태아 혈청을 첨가해준다. 혈청의 경우 프리온이나 바이러스에 의한 오염이 있을 수 있고, 제품간 품질의 차이가 날 수 있다. 게다가 호주나 뉴질랜드에서 유래된 고품질 혈청의 경우 매우 비싸기 때문에 제조 비용이 증가된다. In the case of cell culture, fetal serum is usually added as a growth factor. Serum may be contaminated by prions or viruses, and quality differences may occur between products. In addition, high quality serum derived from Australia or New Zealand is very expensive and increases manufacturing costs.
Vero 세포주는 다양한 바이러스가 증식할 수 있는 확립 세포주이다. 그러나 Vero 세포주는 표면에 대한 부착성이 매우 강하기 때문에 대량 배양을 위해서는 매우 넓은 면적의 배양 용기나 담체가 필요하고 이는 많은 비용이 든다. 따라서 시설 또는 담체에 대한 비용이 막대하며 배양용기에 부착된 세포를 제거하는 단계가 필요하며 이로 인해 세포의 손실 및 손상이 있다. 따라서 저렴하고 안전하게 동물세포 배양을 통한 백신을 제조하기 위해서는 무혈청 및 부유 배양이 가능한 세포주가 필요하다. The Vero cell line is an established cell line capable of multiplying various viruses. However, since the Vero cell line is highly adherent to the surface, a large-scale culture container or carrier is required for mass culture, which is costly. Thus, the cost of the facility or carrier is enormous and a step of removing cells attached to the culture vessel is required, which results in cell loss and damage. Therefore, in order to produce a vaccine through animal cell culture inexpensively and safely, a cell line capable of serum-free and floating culture is required.
따라서 본 발명이 해결하고자 하는 과제는 무혈청 배양 및 부유 배양이 가능하다는 장점이 있고, 백신용 바이러스 증식에 사용될 수 있는 Vero 세포주를 제공하는 것이다. 또한, 본 발명은 상기 세포주를 이용하여 바이러스, 특히 로타 바이러스를 생산하는 방법을 제공하는 것이다.Therefore, a problem to be solved by the present invention is to provide a Vero cell line which can be used for serum-free virus propagation, and has the advantage of being capable of serum-free culture and suspension culture. The present invention also provides a method for producing viruses, particularly rotavirus, using the cell lines.
상기 과제를 해결하기 위하여, 본 발명은 ATCC CCL-81 혹은 WHO에서 분양 받은 Vero (African Green Monkey Kidney) 세포로부터 유도되고, 세포 성장을 위하여 혈청을 필요로 하지 않으며, 부착을 위한 담체가 필요 없이 부유 배양이 가능한 특정 Vero 세포주를 제공한다.In order to solve the above-mentioned problems, the present invention relates to a method for producing a cell line derived from ATCC CCL-81 or Vero (African Green Monkey Kidney) cells distributed by WHO, Specific Vero cell lines capable of culturing.
상기 바람직한 특성인 무혈청 배양 및 부유 배양이 가능한 Vero 세포주는 다음과 같은 단계를 거쳐 제조될 수 있다. 구체적으로, S1) Vero 세포주를 단계적으로 혈청 비율을 줄여가며 무혈청 배지에 적응시켜 무혈청 배양이 가능한 세포주를 제조하고, S2) 무혈청 배양이 가능한 세포주를 부유 배양을 위한 두 종류의 배양 용기(Ultra-low attachment 플라스크, 스피너(spinner) 플라스크)를 이용하여 부유 배양에 단계적으로 적응시켜 부유 배양이 가능한 세포주를 선별하는 방법으로 제조할 수 있다. Vero cell lines capable of serum-free culture and suspension culture, which are the above-mentioned preferable characteristics, can be produced through the following steps. Specifically, S1) Vero cell line was stepwise reduced to a serum-free medium to prepare a cell line capable of serum-free culture. S2) A cell line capable of serum-free culture was prepared from two types of culture containers An ultra-low attachment flask, or a spinner flask) to select a cell line capable of suspension culture.
더욱 자세하게는, (a) ATCC CCL-81 혹은 WHO에서 분양 받은 Vero 세포를 준비하는 단계; (b) 상기 Vero 세포를, 화학적으로 정의된 무혈청 배지에서 생장하도록 적응시키는 단계; 및 (c) 단계 (b)에서 적응된 부착성인 상기 Vero 세포를, 화학적으로 정의된 무혈청 배지 중에서 두 단계에 걸쳐 부유배양 적응을 유도하여 최종적으로 스피너(spinner) 플라스크에서 부유 상태로 생장하도록 적응시킨 Vero 세포를 제조하는 방법 및 이러한 방법을 통해 얻어진 무혈청 및 부유 배양이 가능한 Vero 세포주를 제공한다.(A) preparing Vero cells distributed from ATCC CCL-81 or WHO; (b) adapting said Vero cells to grow in chemically defined serum-free medium; And (c) adapting the adhered Vero cells in step (b) to induce suspension culture adaptation over two steps in a chemically defined serum-free medium to finally adapt to growth in a floating state in a spinner flask And a Vero cell line capable of serum-free and suspension culture obtained through such a method.
바람직하게, 상기 방법을 통하여 신규 확립된 무혈청 및 부유 배양이 가능한 Vero 세포주는 Vero Sky7458 (기탁번호: KCLRF-BP-00377)이며, 한국세포주은행에 2016년 10월 27일자로 기탁하였다.Preferably, the Vero SK7458 (Accession No .: KCLRF-BP-00377), a newly established serum-free and floating cultureable Vero cell line, is deposited at the Korean Cell Line Bank on Oct. 27,
본 발명에 있어, 용어 "무혈청 배지"는 혈청이 실질적으로 첨가되지 않은, 본 발명에 따른 확립세포주가 배양될 수 있는 배지를 의미하며, 용어 "실질적으로 첨가되지 않은"은 혈청을 0.5 (v/v) % 이하 포함하는 것을 의미하며, 바람직하게는 0.1 (v/v) % 이하, 더욱 바람직하게는 0.01 (v/v) % 이하, 가장 바람직하게는 전혀 포함하지 않는 것을 의미한다.In the present invention, the term "serum-free medium" means a medium in which the established cell line according to the present invention can be cultured in which substantially no serum is added, and the term " (v / v)%, more preferably not more than 0.01 (v / v)%, and most preferably not at all.
본 발명은 또한 본 발명에 따른 세포주를 이용하여 백신용 바이러스를 제조하는 방법을 제공한다. 본 발명에 따른 세포주를 이용하여 증식될 수 있는 바이러스는, 예를 들어, 로타 바이러스, 뎅기 바이러스, 인플루엔자 바이러스, 홍역바이러스, 일본뇌염바이러스, 이하선염바이러스, 풍진바이러스, 폴리오바이러스, HSV-1, HSV-2, 광견병바이러스, RS바이러스, 레오바이러스 3형, 황열바이러스, 파보바이러스, 콕사키바이러스, 아데노바이러스 1형 내지 47형, 라사바이러스 및 백시니아바이러스 등이 있으며, 본 발명에 따른 세포주는 이러한 바이러스들 중 로타 바이러스의 생산에 더욱 적합하다. The present invention also provides a method for producing a vaccine virus using a cell line according to the present invention. Viruses which can be propagated using the cell line according to the present invention include viruses such as rotavirus, dengue virus, influenza virus, measles virus, Japanese encephalitis virus, mumps virus, rubella virus, poliovirus, HSV- 2, rabies virus, RS virus, reovirus type 3, yellow fever virus, parvovirus, cocksuck virus, adenovirus type 1 to 47 type, lasa virus and vaccinia virus. Lt; RTI ID = 0.0 > rotavirus. ≪ / RTI >
보다 구체적으로, 본 발명은 (a) 본 발명에 따른 Vero 세포를 이용하여 1×105 내지 9×105 cells/㎖의 접종 농도, 바람직하게는 약 5×105개 cells/㎖의 접종 농도로 무혈청 세포 배양 배지를 접종하는 단계; (b) 40 내지 80 rpm의 교반 속도, 바람직하게는 약 60 rpm의 교반 속도, 약 6.5-7.5의 pH의 배양 조건을 유지 하면서 상기 세포를 배양하는 단계를 포함하여, 스피너(spinnre) 플라스크에서 상기 Vero 세포를 1.0 ×106 내지 2.0×106개 수준 cells/㎖의 세포 밀도, 바람직하게는 약 1.5×106개 수준 cells/㎖의 세포 밀도로 증식시키는 단계; (c) 상기 증식된 Vero 세포를 로타바이러스로 감염시키는 단계; (d) 로타바이러스의 복제를 허용하는 조건 하에 상기 감염된 증식 Vero 세포를 배양하는 단계; 및 (e) 세포 배양 조성물로부터 로타바이러스를 분리하는 단계를 포함하는 것을 특징으로 하는 세포 배양물에서 로타바이러스를 생산하는 방법을 제공하며, 바람직하게, 본 발명의 이러한 방법은 상기 단계 (b) 동안에는 상기 세포 배양물에 신선한 배지의 추가 또는 배지를 일부 제거하여 신선한 배지로 대체하는 단계를 추가로 포함한다.More particularly, the present invention relates to a method for producing a vaccine composition comprising: (a) using Vero cells according to the present invention, an inoculation concentration of 1 x 10 5 to 9 x 10 5 cells / ml, preferably an inoculation concentration of about 5 x 10 5 cells / Inoculated with a serum-free cell culture medium; (b) culturing said cells while maintaining culture conditions at a stirring rate of 40-80 rpm, preferably a stirring rate of about 60 rpm, and a pH of about 6.5-7.5, in a spinner flask, Vero cells were seeded at 1.0 x 10 < 6 > Growing at a cell density of 2.0 x 10 6 cells / ml, preferably at a cell density of about 1.5 x 10 6 cells / ml; (c) infecting the proliferated Vero cells with rotavirus; (d) culturing said infected proliferating Vero cells under conditions permitting replication of rotavirus; And (e) separating the rotavirus from the cell culture composition. Preferably, the method of the present invention is characterized in that during the step (b) Adding the fresh medium to the cell culture or partially removing the medium and replacing with fresh medium.
본 발명은 또한 본 발명에 따른 방법에 의해 제조된 바이러스 또는 바이러스 항원을 제공하며, 이러한 바이러스 항원을 포함하는 면역원성 약학 조성물을 제공한다.The present invention also provides a virus or viral antigen produced by the method according to the present invention, and provides an immunogenic pharmaceutical composition comprising such a viral antigen.
본 발명은 무혈청 배양 및 부유 배양이 가능하며 바이러스 증식에 효율적으로 사용될 수 있는 신규 Vero 세포주를 제공한다. 본 발명은 또한 이러한 세포주를 이용하여 바이러스, 특히 로타바이러스를 생산하는 방법을 제공한다.The present invention provides a novel Vero cell line which can be used for serum-free culture and suspension culture and can be effectively used for virus propagation. The present invention also provides methods for producing viruses, particularly rotaviruses, using such cell lines.
본 명세서에 첨부되는 다음의 도면들은 본 발명의 바람직한 실시예를 예시하는 것이며, 전술한 발명의 내용과 함께 본 발명의 기술사상을 더욱 이해시키는 역할을 하는 것이므로, 본 발명은 그러한 도면에 기재된 사항에만 한정되어 해석되어서는 아니 된다.
도 1은 본 발명에 따른 무혈청 Vero 세포주들을 Ultra-low attachment 플라스크에서 부유 배양한 결과로, 계대에 따른 세포 농도 증가 형태를 보여준다.
도 2는 본 발명에 따른 Vero 세포주들을 스피너(spinner) 플라스크에서 부유 배양한 결과로, 계대에 따른 세포 농도 증가 형태를 보여준다.
도 3은 본 발명에 따른 Vero 세포주에서 증식된 로타바이러스 시료의 plaque assay 결과이다.BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments of the invention and, together with the description of the invention, It should not be construed as limited.
FIG. 1 shows the results of suspension culture of serum-free Vero cell lines according to the present invention in an ultra-low attachment flask.
FIG. 2 shows the morphology of increased cell concentration of the Vero cell lines according to the present invention as a result of suspension culture in a spinner flask.
FIG. 3 is a plaque assay result of a rotavirus sample proliferated in a Vero cell line according to the present invention.
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석되어서는 안 된다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.
<실시예 1> 무혈청 부유배양 Vero 세포주의 제조<Example 1> Preparation of Vero cell line without serum suspension
Vero 세포주 CCL-81는 ATCC에서 분양 받았다. T-75 플라스크 내 10% 혈청을 포함한 EMEM 배지에서 배양 온도 37℃, 5% CO2 조건에서 배양하였다. 상기 세포를 확장한 후 상기 배지에 무혈청 배지를 50% 포함한 배지에 배양을 하고 세포의 이상이 없는지 확인하였다. 세포의 성장에 이상이 없을 경우 무혈청 배지 75% 포함한 배지에서 배양을 해주고 상기 방법을 반복하여 100% 무혈청 배지에 적응된 세포주를 확보하였다. 무혈청 배양에 사용된 배지는 예를 들면 Sky-FM03 (Lonza) EX-CELL MDCK (Sigma), VP-SFM (Gibco) 등을 들 수 있다. The Vero cell line CCL-81 was distributed at the ATCC. The cells were cultured in EMEM medium containing 10% serum in a T-75 flask at a culture temperature of 37 ° C and 5% CO 2 . After the cells were expanded, the medium was cultured in a medium containing 50% serum-free medium to confirm that there was no abnormality in the cells. When there was no abnormality in the growth of the cells, the cells were cultured in a medium containing 75% of serum-free medium, and the above procedure was repeated to obtain a cell line adapted to 100% serum-free medium. Examples of the medium used for serum-free culture include Sky-FM03 (Lonza) EX-CELL MDCK (Sigma) and VP-SFM (Gibco).
무혈청 배지에 적응된 세포주를 T-플라스크에서 충분히 확장한 후 Ultra-low attachment T-플라스크에서 부유 배양 적응을 시작하였다. 배양 온도 37℃, 5% CO2 조건에서 배양하였다. 배양 배지의 pH가 낮아지거나 세포가 일정 수준이상으로 자랐을 경우 배지 교환을 해주거나 계대 배양을 해주었다. 2개월의 부유 배양 적응을 거친 후 안정적인 세포성장이 확인 되었다. 세포의 생존율은 90% 이상임을 확인하였다. 이후 상기 세포주를 충분히 확장하여 스피너(spinner) 플라스크(Corning)에서 부유 배양 적응을 시작하였다. 교반속도는 60rpm이고 배양 온도 37℃, 5% CO2 조건에서 배양하였다. 배양 배지의 pH가 낮아지거나 세포가 일정 수준이상으로 자랐을 경우 배지 교환을 해주거나 계대 배양을 해주었다. 3~6개월의 부유 배양 적응을 거친 후 세포 농도가 접종 3-4일 차에 약 1.3 ~ 1.5×106 cells/ml 수준이 되었다. 세포의 생존율은 90% 이상임을 확인하였다. 부유 배양에 사용된 배지는 예를 들면 Sky-FM03 (Lonza) EX-CELL MDSK (Sigma), VP-SFM (Gibco) 등을 들 수 있다. 상기 방법으로 무혈청 배양 및 부유 배양이 가능한 Vero 세포주를 확보하였으며 Vero Sky7458로 명명하였다.Cell lines adapted to serum-free medium were fully expanded in T-flasks and then adapted for suspension culture in Ultra-low attachment T-flasks. And cultured at 37 ° C and 5% CO 2 . When the pH of the culture medium was lowered or the cells were grown above a certain level, the medium was exchanged or subcultured. After 2 months suspension culture adaptation, stable cell growth was confirmed. The survival rate of the cells was 90% or more. Subsequently, the cell line was fully expanded and the suspension culture adaptation was started in a spinner flask (Corning). The agitation speed was 60 rpm and incubation was carried out at a culture temperature of 37 캜 and 5% CO 2 . When the pH of the culture medium was lowered or the cells were grown above a certain level, the medium was exchanged or subcultured. After 3 to 6 months of suspension culture adaptation, the cell concentration was about 1.3 to 1.5 × 10 6 cells / ml at 3-4 days of inoculation. The survival rate of the cells was 90% or more. Examples of the medium used for suspension culture include Sky-FM03 (Lonza) EX-CELL MDSK (Sigma), VP-SFM (Gibco) and the like. Vero cell line, which can be used for serum-free culture and suspension culture, was obtained by this method and named as Vero Sky7458.
<실시예 2> 세포주의 증식성 평가<Example 2> Evaluation of cell proliferation
무혈청 배양 Vero 세포주를 하기 표 1의 조건 하에서 배양하여 그 증식성을 평가하였다. 대조군으로서 Microcarrier를 이용하여 배양한 무혈청 적응 Vero 세포주(VP-SFM)를 사용하였다.Serum-free culture Vero cell lines were cultured under the conditions shown in Table 1, and their proliferation was evaluated. A serum-free adaptive Vero cell line (VP-SFM) cultured using a microcarrier was used as a control.
3일 내지 4일 경과 후 세포를 회수하여 1200rpm, 5분간 원심분리 하고 5.0×105 cells/ml 로 계대 배양 한다. After 3 to 4 days, the cells were collected, centrifuged at 1200 rpm for 5 minutes, and subcultured at 5.0 × 10 5 cells / ml.
본 발명에서 만들어진 무혈청 Vero 세포주들은 혈청 배지에서 성장하는 Vero 세포주와 비교했을 때 동등한 수준의 세포 성장율을 보였다. The serum-free Vero cell lines produced in the present invention showed comparable levels of cell growth compared to Vero cell lines grown in serum medium.
<실시예 3> 세포주의 증식 형태 및 계대 안정성 평가≪ Example 3 > Evaluation of propagation morphology and stability of cell line
무혈청 배양 및 부유배양 적응된 세포주에 대하여 스피너(spinner) 플라스크에서 연속적으로 배양하여 세포 증식 형태 및 계대에 따른 안정성을 확인하였다. 배양 개시 세포 농도는 약 5×105 cells/ml로 하였고 배양 약 3~4일 경과 후 세포 농도는 약 1.3~1.5×106 cells/ml 수준까지 도달하였다. 배양 조건은 하기와 같다. 그 결과를 도 2에 나타내었다.The cell cultures adapted to serum-free culture and suspension culture were continuously cultured in a spinner flask to confirm the stability depending on cell growth pattern and passage. The culture initiation cell concentration was about 5 × 10 5 cells / ml. After about 3 to 4 days, the cell concentration reached about 1.3 to 1.5 × 10 6 cells / ml. The culture conditions are as follows. The results are shown in Fig.
개시 세포 농도: 5×105 cells/mlInitiation cell concentration: 5 × 10 5 cells / ml
배양 규모: 125ml spinner flask Culture scale: 125ml spinner flask
Spinner 회전수: 60 rpmSpinner RPM: 60 rpm
배양 조건: 37℃, 5% CO2, 습윤하Culture conditions: 37 ° C, 5% CO 2 , wet
계대 조건: 배양 3~4일 경과 후Stage conditions: After 3 to 4 days of incubation
<실시예 4> 바이러스 증식 평가Example 4 Evaluation of Virus Growth
본 발명의 상기 Vero 세포들을 이용하여 부유 배양 조건에서 바이러스를 증식시켰다. 본 실험에 사용된 바이러스는 로타바이러스 (RV : ATCC VR-2493)였으며, 배양 조건은 하기와 같다.The Vero cells of the present invention were used to propagate the virus in suspension culture conditions. The virus used in this experiment was rotavirus (RV: ATCC VR-2493), and the culture conditions were as follows.
세포 농도: 1.5×106 cells/mlCell concentration: 1.5 × 10 6 cells / ml
배양 규모: 125ml spinner flask Culture scale: 125ml spinner flask
Spinner 회전수: 40 rpmSpinner RPM: 40 rpm
배양 조건: 37℃, 5% CO2, 습윤하Culture conditions: 37 ° C, 5% CO 2 , wet
배양 기간: 5일Cultivation period: 5 days
세포주를 이용한 바이러스 배양 시 감염을 위하여 트립신을 배양배지에 포함하였다. 로타 바이러스의 역가 측정은 plaque assay를 수행하여 측정하였다. 5일간 배양 후 배양 상층액을 회수하고 바이러스 역가를 측정하였다. 측정 결과를 하기 표 2에 나타내었다. 표 2에 나타난 바와 같이 1.0×107 PFU/mL 이상의 역가(titer)를 나타냈다. 이러한 바이러스 증식은 무혈청 배지에서 배양된 Vero 세포의 것과 유사한 수준이었다. 따라서, 본 발명의 Vero 세포주가 무혈청 및 부유 배양 상태에서 바이러스를 효율적으로 제조할 수 있음이 판명되었다.When the virus was cultured using the cell line, trypsin was included in the culture medium for infection. The titers of rotavirus were measured by plaque assay. After incubation for 5 days, culture supernatant was collected and viral titers were measured. The measurement results are shown in Table 2 below. As shown in Table 2, the titer was 1.0 x 10 7 PFU / mL or more. These virus proliferation was similar to that of Vero cells cultured in serum-free medium. Thus, it has been found that the Vero cell line of the present invention can efficiently produce viruses in serum-free and suspended culture conditions.
(PFU/mL)Virus titer
(PFU / mL)
Claims (9)
(b) 40 내지 80 rpm의 교반 속도, 6.5-7.5의 pH 배양 조건을 유지 하면서 상기 세포를 배양하는 단계를 포함하여, 스피너(spinner)플라스크에서 상기 Vero 세포를 1.0 ×106 내지 2.0×106개 수준 cells/㎖의 세포 밀도로 증식시키는 단계;
(c) 상기 증식된 베로(Vero) 세포를 로타바이러스로 감염시키는 단계;
(d) 로타바이러스의 복제를 허용하는 조건 하에 상기 감염된 증식 Vero 세포를 배양하는 단계; 및
(e) 세포 배양 조성물로부터 로타바이러스를 분리하는 단계
를 포함하는 것을 특징으로 하는 백신용 로타바이러스를 생산하는 방법.(a) inoculating a serum-free cell culture medium with the inoculation concentration of 1 × 10 5 to 9 × 10 5 cells / ml using the Vero cells of (1) or (2);
(b) incubating said Vero cells in a spinner flask at a concentration of from 1.0 x 10 < 6 > to 10 x 10 < -6 > in a spinner flask, comprising culturing said cells while maintaining a stirring rate of 40-80 rpm, Growing at a cell density of 2.0 x 106 cells / ml;
(c) infecting the proliferated Vero cells with rotavirus;
(d) culturing said infected proliferating Vero cells under conditions permitting replication of rotavirus; And
(e) isolating rotavirus from the cell culture composition
≪ / RTI > wherein the method comprises the steps of:
(b) 상기 베로(Vero) 세포를 무혈청 배지에서 생장하도록 적응시키는 단계; 및
(c) 단계 (b)에서 적응된 부착성인 상기 베로(Vero) 세포를 무혈청 배지 중에서 부착을 위한 담체를 필요로 하지 않고 부유 상태에서 생장하도록 적응시킨 베로(Vero) 세포를 수득하는 단계를 포함하는,
세포 성장을 위하여 혈청을 필요로 하지 않으며, 부착을 위한 담체가 필요 없이 부유 배양이 가능한 제1항 또는 제2항의 세포주를 제조하는 방법.
(a) preparing a Vero cell deposited as ATCC CCL-81;
(b) adapting the Vero cells to grow in serum-free medium; And
(c) obtaining a Vero cell adapted for attachment in step (b), wherein said Vero cells are adapted to grow in a suspended state in a serum-free medium without the need for a carrier for attachment; doing,
A method for producing a cell line according to claim 1 or 2, which does not require serum for cell growth and is capable of suspension culture without the need for a carrier for attachment.
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| KR1020170080531A KR101831284B1 (en) | 2017-06-26 | 2017-06-26 | Vero Cell Lines Suspension-cultivated Without Serum and Methods for Preparing Vaccine Virus With Those Cell Lines |
| PCT/KR2018/001905 WO2019004556A1 (en) | 2017-06-26 | 2018-02-13 | Vero cell line suspension-cultured in serum-free medium and method for producing vaccine virus using same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021107612A1 (en) | 2019-11-27 | 2021-06-03 | 에스케이바이오사이언스 주식회사 | Novel vero cell line that can be suspension-cultured in serum-free medium, preparation method therefor, and method for preparing viruses for vaccines by using novel cell line |
| CN114540277A (en) * | 2022-03-18 | 2022-05-27 | 杭州荣泽生物科技集团有限公司 | Serum-free medium for culturing Vero cells and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110904025A (en) * | 2019-10-08 | 2020-03-24 | 上海源培生物科技股份有限公司 | Serum-free suspension domestication method of Vero cells |
| CN114410592B (en) * | 2021-12-30 | 2024-05-24 | 苏州百源基因技术有限公司 | Rotavirus culture medium and application thereof |
| CN114209822B (en) * | 2022-01-23 | 2025-03-18 | 中牧实业股份有限公司 | Adjuvant for porcine Japanese encephalitis inactivated vaccine composition and its composition and application |
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| US20090203112A1 (en) * | 2005-10-04 | 2009-08-13 | Ricardo Kratje | Vero cell line which is adapted to grow in suspension |
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| US6656719B1 (en) * | 1997-10-27 | 2003-12-02 | Merck & Co., Inc. | Serum-free, low-protein media for rotavirus vaccine production |
| GB201011502D0 (en) * | 2010-07-08 | 2010-08-25 | Glaxosmithkline Biolog Sa | Novel process |
| WO2012033236A1 (en) * | 2010-09-06 | 2012-03-15 | 에스케이케미칼 주식회사 | Mdck cell line adapted to serum-free culture and suspension culture, and method for preparing viruses for vaccine using cells thereof |
| CN106834239A (en) * | 2017-01-20 | 2017-06-13 | 江苏中慧元通生物科技有限公司 | A kind of serum-free Vero cells prepare the method and serum-free hydrophobia vaccine product of rabies vacciness stoste |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20090203112A1 (en) * | 2005-10-04 | 2009-08-13 | Ricardo Kratje | Vero cell line which is adapted to grow in suspension |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021107612A1 (en) | 2019-11-27 | 2021-06-03 | 에스케이바이오사이언스 주식회사 | Novel vero cell line that can be suspension-cultured in serum-free medium, preparation method therefor, and method for preparing viruses for vaccines by using novel cell line |
| KR20210065697A (en) | 2019-11-27 | 2021-06-04 | 에스케이바이오사이언스(주) | New Vero cell lines capable of suspension culture in serum-free media, and method for preparing the cell lines, and method for producing vaccine virus using the cell lines |
| JP2023505101A (en) * | 2019-11-27 | 2023-02-08 | エスケー バイオサイエンス カンパニー リミテッド | Novel Vero Cell Strain Capable of Suspension Culture in Serum-Free Medium, Method for Producing Same, and Method for Producing Vaccine Virus Using the Novel Cell Strain |
| JP7688639B2 (en) | 2019-11-27 | 2025-06-04 | エスケー バイオサイエンス カンパニー リミテッド | A novel Vero cell line capable of being cultured in suspension in serum-free medium, its production method, and a method for producing a vaccine virus using the novel cell line |
| CN114540277A (en) * | 2022-03-18 | 2022-05-27 | 杭州荣泽生物科技集团有限公司 | Serum-free medium for culturing Vero cells and preparation method thereof |
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