Veroe6 tmprss2 cell

Manufactured by Japanese Collection of Research Bioresources Cell Bank

Sourced in Japan, United States

VeroE6/TMPRSS2 cells are a cell line derived from African green monkey kidney cells, engineered to express the TMPRSS2 protein. The cells are maintained in culture and can be used for various research applications.

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Lab products found in correlation

Veroe6, by Japanese Collection of Research Bioresources Cell Bank (4 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions) Amphotericin b, by Bristol-Myers Squibb (3 mentions) Kanamycin, by Meiji Seika Pharma (3 mentions) Dulbecco modified eagle medium (dmem), by Nacalai Tesque (3 mentions) Vero e6 cell, by American Type Culture Collection (3 mentions) Penicillin streptomycin, by Merck Group (3 mentions) Crystal violet solution, by Merck Group (3 mentions) Formalin, by Fujifilm (3 mentions) Fetal bovine serum (fbs), by Biowest (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Doxycycline hydrochloride, by Merck Group (3 mentions) Dmem low glucose, by Merck Group (3 mentions) Lenti x 293t cells, by Takara Bio (2 mentions) Non essential amino acids (neaa), by Corning (2 mentions) L glutamine, by Corning (2 mentions) Sodium pyruvate, by Corning (2 mentions) Antibiotic and antimycotic, by Corning (2 mentions) Hek293t cell, by American Type Culture Collection (2 mentions)

Spelling variants of this product

VeroE6/TMPRSS2 (21 citations) VeroE6 (8 citations) VeroE6 cells (6 citations) VeroE6/TMPRSS2 cells (JCRB1819) (3 citations) VeroE6/TMPRSS2 cell line (3 citations)

55 protocols using veroe6 tmprss2 cell

SARS-CoV-2 Neutralizing Antibody Assay

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The analysis of the SARS-CoV-2 neutralizing antibody responses was performed in a VeroE6-TMPRSS2 cell (JCRB Cell Bank)^{32 (link)} culture assay in presence of live virus. Pre-incubated mixtures (at different dilution ratios) of NHP heat-inactivated sera (56 °C for 30 min) and SARS-CoV-2 virus (strain SK-BMC5/2020 supplied through the European Virus Archive - Global [EVAg] platform) were placed on plated cells. A reference standard control was included (WHO International Standard First WHO International Standard for anti-SARS-CoV-2 human immunoglobulin NIBSC code 20/136, corresponding to pooled plasma obtained from eleven convalescent individuals recovered from SARS-CoV-2 infection). Data acquisition was performed on an Operetta reader (Perkin Elmer), using the colorimetric CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay following the manufacturer’s protocol (Promega, ref. #G5430). Using XLfit®, the corresponding Neutralizing titers (NT₅₀) were calculated using the nonlinear regression model, expressed as the inverse of the serum dilution that inhibited 50% of infection (lower limit of quantification: 5; NIBSC 20/136 NT₅₀: 21).

Galhaut M., Lundberg U., Marlin R., Schlegl R., Seidel S., Bartuschka U., Heindl-Wruss J., Relouzat F., Langlois S., Dereuddre-Bosquet N., Morin J., Galpin-Lebreau M., Gallouët A.S., Gros W., Naninck T., Pascal Q., Chapon C., Mouchain K., Fichet G., Lemaitre J., Cavarelli M., Contreras V., Legrand N., Meinke A, & Le Grand R. (2024). Immunogenicity and efficacy of VLA2001 vaccine against SARS-CoV-2 infection in male cynomolgus macaques. Communications Medicine, 4, 62.

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Cell Culture Methodology for SARS-CoV-2 Research

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The cell lines used in this study were available in our laboratory as previously described 24 (link). Caco2 cell was acquired from ATCC (ATCC HTB-37) and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Amarillo, Texas, USA) according to supplier's manual and guidance. VeroE6-TMPRSS2 cell was acquired from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB1819) and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Amarillo, Texas, USA) according to instructions. All cell lines that were used in this study underwent mycoplasma testing regularly and were cultivated in mycoplasma-free environment.

Yuen T.T., Chan J.F., Yan B., Shum C.C., Liu Y., Shuai H., Hou Y., Huang X., Hu B., Chai Y., Yoon C., Zhu T., Liu H., Shi J., Zhang J., Cai J.P., Zhang A.J., Zhou J., Yin F., Yuan S., Zhang B.Z, & Chu H. (2022). Targeting ACLY efficiently inhibits SARS-CoV-2 replication. International Journal of Biological Sciences, 18(12), 4714-4730.

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SARS-CoV-2 Virus Isolation and Titration

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JPN/TY/WK‐521 strain of SARS‐CoV‐2 was provided by the National Institute of Infectious Diseases (Tokyo, Japan). VeroE6/TMPRSS2 cell was obtained from the Japanese Collection of Research Bioresources (Osaka, Japan, Cell number: JCRB1819). SARS‐CoV‐2 was inoculated to the VeroE6/TMPRSS2 cells cultured in Dulbecco's modified Eagle's medium (Nissui Pharmaceutica, Tokyo, Japan) supplemented with 1% (v/v) fetal bovine serum, 2 mm l‐glutamine (Fujifilm Wako Pure Chemical, Osaka, Japan), 100 μg·mL⁻¹ kanamycin (Meiji Seika Pharma, Tokyo, Japan), and 2 μg·mL⁻¹ amphotericin B (Bristol‐Myers Squibb, New York, NY, USA). After 3 days, the cell culture supernatant was collected and used as a SARS‐CoV‐2 solution. Based on the degree of cytopathic effect observed on VeroE6/TMPRSS2 cells, the viral titer of SARS‐CoV‐2 solution was determined to be 7.0 log₁₀ 50% tissue culture infective dose (TCID₅₀)·mL⁻¹.

Nakazawa D., Takeda Y., Kanda M., Tomaru U., Ogawa H., Kudo T., Shiratori‐Aso S., Watanabe‐Kusunoki K., Ueda Y., Miyoshi A., Hattanda F., Nishio S., Uozumi R., Ishizu A, & Atsumi T. (2022). Transcriptional dynamics of granulocytes in direct response to incubation with SARS‐CoV‐2. FEBS Open Bio, 13(1), 60-71.

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Evaluating Antiviral Agents Against SARS-CoV-2

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VeroE6 cells and TMPRSS2-overexpressing VeroE6 (VeroE6^TMPRSS2) cells were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). VeroE6 cells were maintained in Dulbecco’s modified Eagle’s medium (d-MEM) supplemented with 10% fetal bovine serum (FCS), 100 μg/ml of penicillin, and 100 μg/ml of streptomycin. VeroE6^TMPRSS2 cells were maintained in d-MEM as mentioned above in the presence of 1 mg/ml of G418. SARS-CoV-2 strain JPN/TY/WK-521 (SARS-CoV-2^WK-521) was obtained from the National Institute of Infectious Diseases (Tokyo, Japan).
The antiviral agents lopinavir (Sigma-Aldrich, St. Louis, MO); nelfinavir, nafamostat, hydroxy chloroquine, and nitazoxanide (Tokyo Chemical Industry, Tokyo, Japan); favipiravir (MedChemExpress, Monmouth Junction, NJ); and chloroquine (Selleck, Sylvanfield Drive, Houston, TX) were purchased. Remdesivir was obtained from Clifford Lane, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. GRL-0820 and GRL-0920 were synthesized by A. K. Ghosh. Each compound except remdesivir was dissolved in DMSO at 20 mM, and remdesivir was prepared with saline solution at 5 mM concentrations as stock solutions.

Hattori S.I., Higshi-Kuwata N., Raghavaiah J., Das D., Bulut H., Davis D.A., Takamatsu Y., Matsuda K., Takamune N., Kishimoto N., Okamura T., Misumi S., Yarchoan R., Maeda K., Ghosh A.K, & Mitsuya H. (2020). GRL-0920, an Indole Chloropyridinyl Ester, Completely Blocks SARS-CoV-2 Infection. mBio, 11(4), e01833-20.

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Gamma SARS-CoV-2 Mouse Adaptation Protocol

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The Gamma strain (hCoV-19/Japan/TY7-503/2021) of SARS-CoV-2 was obtained from the National Institute of Infectious Diseases (Tokyo, Japan). The virus was expanded in VeroE6/TMPRSS2 cells (JCRB Cell Bank, Osaka, Japan) and stored at −80 °C until use. VeroE6/TMPRSS2 cells were cultured in DMEM (Nacalai Tesque, Kyoto, Japan) containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin. Mice were intranasally challenged with MA10, a mouse-adapted SARS-CoV-2. MA10 was generated using a circular polymerase extension reaction as described previously (53 (link)). The SARS-CoV-2 NIID strain (2019-nCoV_Japan_TY_WK-5212020) served as the backbone of MA10. MA10 contains seven mutations, which were introduced adaptively into SARS-CoV-2 during serial passages in BALB/c mice (54 (link)). Each experiment with live SARS-CoV-2 was performed within a biosafety level 3 facility at Osaka University, adhering to stringent guidelines.

Tokunoh N., Tamiya S., Watanabe M., Okamoto T., Anindita J., Tanaka H., Ono C., Hirai T., Akita H., Matsuura Y, & Yoshioka Y. (2023). A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice. Frontiers in Immunology, 14, 1224634.

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SARS-CoV-2 Strain Propagation and Pseudovirus Generation

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Transmembrane serine protease 2 (TMPRSS-2)-expressing VeroE6 (VeroE6/TMPRSS2) cells [17] (link) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) and maintained in culture medium of Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% heat inactivated fetal bovine serum (FBS) and 1 mg/mL gentamicin (Genticin). SARS-CoV-2 JPN/TY/WK-521 (WK-521, accession no. EPI_ISL_408667), which was isolated from the throat swab of a traveler who had returned from Wuhan on January 31, 2020, was provided by the National Institute of Infectious Diseases (NIID), Japan [17] (link). SARS-CoV-2 JPN/TY/38-873 (Omicron, accession no. EPI_ISL_7418017) was also provided by NIID, Japan. The WK-521 and Omicron strains were propagated on VeroE6/TMPRSS2 cells and virus stocks prepared, which were titered by tissue culture infectious dose 50 (TCID₅₀) assays using VeroE6/TMPRSS2 cells. Lenti-X 293 T cells were purchased from Takara Bio (Shiga, Japan) and used to generate the lentivirus-based D614G, Alpha, Beta, Gamma, Delta, and Omicron pseudoviruses [18] (link). Details on the pseudoviruses are provided in the Supplementary Material.

Hashimoto M., Nagata N., Homma T., Maeda H., Dohi K., Seki N.M., Yoshihara K., Iwata-Yoshikawa N., Shiwa-Sudo N., Sakai Y., Shirakura M., Kishida N., Arita T., Suzuki Y., Watanabe S., Asanuma H., Sonoyama T., Suzuki T., Omoto S, & Hasegawa H. (2022). Immunogenicity and protective efficacy of SARS-CoV-2 recombinant S-protein vaccine S-268019-b in cynomolgus monkeys. Vaccine, 40(31), 4231-4241.

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SARS-CoV-2 Variant Characterization in VeroE6TMPRSS2 Cells

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VeroE6^TMPRSS2 cells^{19 (link)} were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). VeroE6^TMPRSS2 cells were maintained in DMEM supplemented with 10% FCS, 100 µg/ml of penicillin, 100 µg/ml of streptomycin, and 1 mg/ml of G418.
SARS-CoV-2 NCGM-05-2N strain (SCoV2^05-2N) was isolated from nasopharyngeal swabs of a patient with COVID-19, who was admitted to the NCGM hospital^{20 (link)}. hCoV-19/Japan/TKYS02037/2022 (Omicron/BA.2; SARS-CoV-2²⁰³⁷, GISAID Accession ID; EPI_ISL_9397331), hCoV-19/Japan/TKYS14631/2022 (Omicron/BA.5; SARS-CoV-2^TKYS14631, GISAID Accession ID: EPI_ISL_12812500.1), hCoV-19/Japan/TY41-796/2022 (BQ.1.1; SARS-CoV-2^TY41-796, GISAID Accession ID: EPI_ISL_15579783), and hCoV-19/Japan/TY41-795/2022 (XBB; SARS-CoV-2^TY41-795, GISAID Accession ID: EPI_ISL_15669344) were provided from Tokyo Metropolitan Institute of public Health, Japan. hCoV-19/Japan/23-018-P1/2022 (XBB.1.5; SARS-CoV-2^23-018, GISAID Accession ID: EPI_ISL_16889601) was provided by National Institute of Infectious Diseases, Japan. Each variant was confirmed to contain each variant-specific amino acid substitutions.

Amano M., Otsu S., Uemura Y., Ichikawa Y., Matsumoto S., Higashi-Kuwata N., Matsushita S., Shimada S, & Mitsuya H. (2023). Neutralization against Omicron sublineages (BA.2/BA.5/BQ.1.1/XBB/XBB.1.5) in bivalent BNT162b2-vaccinated HCWs with or without risk factors, or following BT infection with Omicron. Scientific Reports, 13, 17404.

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Maintenance of VeroE6/TMPRSS2 Cell Line

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VeroE6/TMPRSS2 cells (JCRB1819, Japanese Collection of Research Bioresources Cell Bank) were maintained in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), 1 mg/mL geneticin, and 100 U/mL penicillin/streptomycin at 37°C supplied with 5% CO₂.

Miyamoto S., Kuroda Y., Kanno T., Ueno A., Shiwa-Sudo N., Iwata-Yoshikawa N., Sakai Y., Nagata N., Arashiro T., Ainai A., Moriyama S., Kishida N., Watanabe S., Nojima K., Seki Y., Mizukami T., Hasegawa H., Ebihara H., Fukushi S., Takahashi Y., Maeda K, & Suzuki T. (2023). Saturation time of exposure interval for cross-neutralization response to SARS-CoV-2: Implications for vaccine dose interval. iScience, 26(5), 106694.

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SARS-CoV-2 Neutralizing Antibody Assay

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Transmembrane protease serine 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6^TMPRSS2) cells (RRID: CVCL_YQ49) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Two SARS-CoV-2 strains, wild-type (PANGO lineage B) and Delta (hCoV-19/Japan/TKYK01734/2021, PANGO lineage B.1.617.2, GISAID Accession ID: EPI_ISL_2080609), were isolated in March 2020 and April 2021, respectively, in Tokyo, Japan, as previously described.^{15 (link)} The SARS-CoV-2 neutralizing activity of sera samples was determined as previously described.^{10 (link),15 (link),16 (link)} All samples used in the present study were evaluated in parallel. High neutralizing activity-confirmed convalescent plasma sample D43^{16 (link)} was used as a reference control to assess the intra-assay deviation.

Terada-Hirashima J., Takamatsu Y., Shimizu Y., Uemura Y., Takeuchi J.S., Tomita N., Matsuda K., Maeda K., Yamamoto S., Fukunaga A., Ohmagari N., Mikami A., Sonoda K., Ujiie M., Mitsuya H, & Sugiura W. (2023). Immunogenicity and safety of single booster dose of KD-414 inactivated COVID-19 vaccine in adults: An open-label, single-center, non-randomized, controlled study in Japan. Human Vaccines & Immunotherapeutics, 19(1), 2193074.

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SARS-CoV-2 Variant Propagation in VeroE6TMPRSS2 Cells

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VeroE6_TMPRSS2 cells^{21 (link)} were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). VeroE6TMPRSS2 cells were maintained in DMEM supplemented with 10% FCS, 100 μg/ml of penicillin, 100 μg/ml of streptomycin, and 1 mg/mL of G418. SARS-CoV-2 NCGM-05–2N strain (SARS-CoV-2_05–2N) was isolated from nasopharyngeal swabs of a patient with COVID-19, who was admitted to the NCGM hospital^{22 (link),23}. Five clinically isolated SARS-CoV-2 mutant strains were used in the current study: two B.1.1.7 (alpha) strains [hCoV-19/Japan/QHN001/2020 (SARS-CoV-2QHN001, GISAID Accession ID; EPI_ISL_804007) and hCoV-19/Japan/QK002/2020 (SARS-CoV-2QK002, G ISAID Accession ID; EPI_ISL_768526)] and a B.1.351 (beta) strain [hCoV-19/Japan/TY8–612-P0/2021 (SARS-CoV-2_TY8–612)] were obtained from National Institute of Infectious Diseases, Tokyo, Japan. A B.1.617.1 (kappa) strain [TKYTK5356_2021 (SARS-CoV-2₅₃₅₆, DDBJ Accession ID; LC633761)] and a B.1.617.1 (beta) strain [hCoV-19/Japan/TKYK01734/2021 (SARS-CoV-2₁₇₃₄, GISAID Accession ID; EPI_ISL_2080609)] were provided from Tokyo Metropolitan Institute of public Health, Tokyo, Japan. Each variant was confirmed to contain each VOC-specific amino acid substitutions before the assays conducted in the present study (vide infra).

Maeda K., Amano M., Uemura Y., Tsuchiya K., Matsushima T., Noda K., Shimizu Y., Fujiwara A., Takamatsu Y., Ichikawa Y., Nishimura H., Kinoshita M., Matsumoto S., Gatanaga H., Yoshimura K., Oka S.I., Mikami A., Sugiura W., Sato T., Yoshida T., Shimada S, & Mitsuya H. (2021). Correlates of Neutralizing/SARS-CoV-2-S1-binding Antibody Response with Adverse Effects and Immune Kinetics in BNT162b2-Vaccinated Individuals. medRxiv.

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