Veroe6 tmprss2

Manufactured by Japanese Collection of Research Bioresources Cell Bank

Sourced in Japan

VeroE6/TMPRSS2 is a cell line that expresses the TMPRSS2 protease. TMPRSS2 is a host cell-derived protein that is important for the entry of SARS-CoV-2 into host cells. This cell line can be used for viral infection and drug screening studies.

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21 protocols using veroe6 tmprss2

Cell Line Maintenance and Characterization

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Calu3, Caco2, 293T, Huh7, and VeroE6 were obtained from the American Type Culture Collection. 293T-ACE2 was obtained from GeneCopoeia (Rockville, Maryland, USA). A549-ACE2 was obtained from Invivogen (San Diego, CA, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Amarillo, Texas, USA) or DMEM/F12 (Gibco) according to the supplier’s instructions (47 (link)). VeroE6-TMPRSS2 was obtained from the Japanese Collection of Research Bioresources Cell Bank and cultured in DMEM. All cell lines used are routinely tested for mycoplasma and are maintained mycoplasma-free.

Chan J.F., Huang X., Hu B., Chai Y., Shi H., Zhu T., Yuen T.T., Liu Y., Liu H., Shi J., Wen L., Shuai H., Hou Y., Yoon C., Cai J.P., Zhang A.J., Zhou J., Yin F., Yuan S., Zhang B.Z., Brindley M.A., Shi Z.L., Yuen K.Y, & Chu H. (2023). Altered host protease determinants for SARS-CoV-2 Omicron. Science Advances, 9(3), eadd3867.

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Cell Lines Used for SARS-CoV-2 Research

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Grivet (Chlorocebus aethiops) kidney epithelial Vero (American Type Culture Collection [ATCC]; CCL-81) and Vero E6 (BEI Resources; NR596), Vero E6/TMPRSS2 (JCRB Cell Bank, Japan; JCRB1819), human colorectal adenocarcinoma Caco-2 (ATCC; HTB-37), HEK293T (ATCC; CRL-3216), human hepatocarcinoma HepG2 (ATCC; #HB-8065), human hepatocarcinoma Huh-7 (a gift from NIH/National Institute of Allergy and Infectious Diseases [NIAID]/Rocky Mountain Laboratories, Hamilton, Montana, USA), human lung adenocarcinoma Calu-3 (ATCC; HTB-55), and human lung carcinoma A549 cells (a gift from University of Rochester Medical Center) were maintained at 37°C and 5% CO₂ in DMEM (Life Technologies) containing 10% heat-inactivated FBS. Primary human hepatocytes from an anonymous 41-year-old woman with colorectal cancer metastasized to the liver were obtained from Liver Center Resources, Pittsburgh Liver Research Center, Pittsburgh, Pennsylvania, USA, and maintained at 37°C and 5% CO₂ in Eagle’s modified essential medium (Gibco).

Bennett R.P., Postnikova E.N., Eaton B.P., Cai Y., Yu S., Smith C.O., Liang J., Zhou H., Kocher G.A., Murphy M.J., Smith H.C, & Kuhn J.H. (2022). Sangivamycin is highly effective against SARS-CoV-2 in vitro and has favorable drug properties. JCI Insight, 7(1), e153165.

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Culturing Human Intestinal Enteroids for COVID-19 Research

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Human intestinal enteroid (HIE) J2 and J3 lines, established from jejunal biopsy specimens of secretor-positive adults (5 (link)), were provided from Baylor College of Medicine under a material transfer agreement. The study protocol was approved by the Review Board of the National Institute of Infectious Diseases in Japan. Wnt3a-producing cells were kindly provided by the Baylor College of Medicine. R-spondin- and Noggin-producing cells were kindly provided by Calvin Kuo (Palo Alto, CA) and Gijs van den Brink (University of Amsterdam, Netherlands), respectively. HIEs were grown as multilobular, 3-dimensional (3D) cultures in Matrigel and were maintained in complete medium with growth factors [CMGF(+)] or IntestiCult organoid growth medium (human) (Veritas) as previously described (5 (link), 10 (link), 19 (link)). Monkey kidney cell line MA104 was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. VeroE6/TMPRSS2 (JCRB1819, VeroE6 cell overexpressing the transmembrane protease, serine 2 [TMPRSS2]) (21 (link)) was purchased from JCRB Cell Bank (Osaka, Japan) and was maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 mg/ml G418 (Nacalai).

Hayashi T., Murakami K., Hirano J., Fujii Y., Yamaoka Y., Ohashi H., Watashi K., Estes M.K, & Muramatsu M. (2021). Dasabuvir Inhibits Human Norovirus Infection in Human Intestinal Enteroids. mSphere, 6(6), e00623-21.

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SARS-CoV-2 Variant Propagation in Vero Cells

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Vero E6 (ATCC CRL-1586), and Vero E6/TMPRSS2 (JCRB Cell Bank, JCRB1819) were cultured in Dulbecco’s modified eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), l-glutamine (2 mM), penicillin (100 U.Ml⁻¹), streptomycin (100 μg.Ml⁻¹) and gentamicin (50 μg.Ml⁻¹). The cell cultures were maintained at 37°C with 5% CO₂. The SARS-CoV-2 isolates used in this study were obtained from residual human anterior nares or nasopharyngeal secretions. The SARS-CoV-2 D614G (B.1 lineage) New York-Ithaca 67-20 (NYI67-20), and Delta (B.1.617.2 lineage) NYI31-21 isolates, were propagated in Vero E6/TMPRSS2 cells, whereas the Omicron BA.1.1 (B.1.1.529) NYI45-21 isolate was propagated in Vero E6 cells. Low passage virus stock (passage 3) were prepared, cleared by centrifugation (2,000 × g for 15 min) and stored at −80°C. The whole genome sequences of the virus stocks were determined to confirm that no mutations occurred during amplification in cell culture. The titers of virus stock were determined by plaque assays, calculated according to the Spearman and Karber method and expressed as plaque-forming units per milliliter (PFU.Ml⁻¹).

Martins M., do Nascimento G.M., Nooruzzaman M., Yuan F., Chen C., Caserta L.C., Miller A.D., Whittaker G.R., Fang Y, & Diel D.G. (2022). The Omicron Variant BA.1.1 Presents a Lower Pathogenicity than B.1 D614G and Delta Variants in a Feline Model of SARS-CoV-2 Infection. Journal of Virology, 96(17), e00961-22.

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SARS-CoV-2 Propagation and Titration in Cell Lines

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Vero cells (ATCC CCL-81), Vero E6 (ATCC CRL-1586), and Vero E6/TMPRSS2 (JCRB Cell Bank; JCRB1819) were cultured in Dulbecco’s modified eagle medium (DMEM), while deer lung (DL; ATCC CRL6195) cells were cultured in minimum essential medium (MEM). Both DMEM and MEM were supplemented with 10% fetal bovine serum (FBS), l-glutamine (2 mM), penicillin (100 U·ml⁻¹), streptomycin (100 μg·ml⁻¹), and gentamicin (50 μg·ml⁻¹). The cell cultures were maintained at 37°C with 5% CO₂. The SARS-CoV-2 isolate TGR/NY/20 obtained from a Malayan tiger naturally infected with SARS-CoV-2 and presenting with respiratory disease compatible with SARS-CoV-2 infection (22 (link)) was propagated in Vero CCL-81 cells. Low-passage virus stocks (passage 4) were prepared, cleared by centrifugation (1,966 × g for 10 min), and stored at −80°C. The endpoint titer was determined by limiting dilution following the Spearman and Karber method. A viral suspension containing 10^6.3 50% tissue culture infectious dose per milliliter (

{TCID}_{50} \cdot {ml}^{- 1}

) was used for all in vitro experiments and fawn inoculations.

Palmer M.V., Martins M., Falkenberg S., Buckley A., Caserta L.C., Mitchell P.K., Cassmann E.D., Rollins A., Zylich N.C., Renshaw R.W., Guarino C., Wagner B., Lager K, & Diel D.G. (2021). Susceptibility of White-Tailed Deer (Odocoileus virginianus) to SARS-CoV-2. Journal of Virology, 95(11), e00083-21.

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Adaptation of SARS-CoV-2 for Mouse Studies

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The maSCV2, originally generated by Ralph Baric (University of North Carolina, Chapel Hill, North Carolina, United States) (45 (link)) was obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources no. NR-55329). The maSCV2 virus was originally generated via infectious clone technology using the sequence of SARS-CoV-2/human/USA/WA-CDC-02982586-001/2020 (WA1 strain) with added mutations in the Spike protein that were predicted to increase binding to murine ACE2 (83 (link)). This virus was further adapted to mice by sequential passage to generate increased virus replication and disease (45 (link)). Working stocks of maSCV2 virus were generated by infecting Vero-E6-TMPRSS2 (Japanese Collection of Research Bioresources Cell Bank no. JCRB1819) cells at a multiplicity of infection (MOI) of 0.01 tissue culture infectious dose 50 (TCID₅₀) per cell in infection media (DMEM; Sigma Aldrich) supplemented with 2.5% filter-sterilized FBS (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco), 1 mM l-glutamine (Gibco), and 1-mM sodium pyruvate (Gibco). Approximately 72 hours after infection, the supernatant fluids were collected, clarified by centrifugation (400g for 10 minutes), and stored in aliquots at –70°C.

Creisher P.S., Perry J.L., Zhong W., Lei J., Mulka K.R., Ryan WH I.I.I., Zhou R., Akin E.H., Liu A., Mitzner W., Burd I., Pekosz A, & Klein S.L. (2023). Adverse outcomes in SARS-CoV-2–infected pregnant mice are gestational age–dependent and resolve with antiviral treatment. The Journal of Clinical Investigation, 133(20), e170687.

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SARS-CoV-2 Propagation in Vero E6 Cells

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Vero E6 (ATCC CRL-1586) and Vero E6/TMPRSS2 (JCRB1819; JCRB Cell Bank) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), l-glutamine (2 mM), penicillin (100 U·ml⁻¹), streptomycin (100 μg·ml⁻¹), and gentamicin (50 μg·ml⁻¹). The cell cultures were maintained at 37°C with 5% CO₂. The SARS-CoV-2 isolate NYI67-20 (B.1 lineage) was propagated in Vero E6 cells. Low-passage-number virus stocks (passage 3) were prepared, cleared by centrifugation (2,000 × g for 15 min), and stored at −80°C. The whole-genome sequence of the virus stock was determined to confirm that no mutations occurred during passages in cell culture. The titer of virus stock was determined according to the Spearman and Karber method and expressed as PFU per milliliter.

Martins M., Fernandes M.H., Joshi L.R, & Diel D.G. (2022). Age-Related Susceptibility of Ferrets to SARS-CoV-2 Infection. Journal of Virology, 96(3), e01455-21.

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Culturing Human Cell Lines for Research

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Human embryonic kidney cell line HEK293T (CRL-3216) and lung adenocarcinoma cells Calu-3 (HTB-55^Tm) were purchased from ATCC. Vero-E6-TMPRSS2 cells were purchased from JCRB Cell Bank. Human embryonic kidney cell line HEK293T and Calu-3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies) and 50 U/mL penicillin-streptomycin (Pen-Strep; Thermo Fisher) at 37°C in a 5% CO₂ atmosphere. Vero-E6-TMPRSS2 cells were cultured in DMEM supplemented with 10% FBS, 1% Pen-Strep, and 1 mg/mL G418 [G418 (Geneticin); InvivoGen].

Lui W.Y., Ong C.P., Cheung P.H., Ye Z.W., Chan C.P., To K.K., Yuen K.S, & Jin D.Y. (2024). Nsp1 facilitates SARS-CoV-2 replication through calcineurin-NFAT signaling. mBio, 15(4), e00392-24.

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Vero E6 Cell Culture with TMPRSS2

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TMPRSS2-expressing Vero E6 (VeroE6/TMPRSS2) cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB1819) and cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) (Biowest, Bradenton, France) and G418 (Nacalai Tesque, Kyoto, Japan). VeroE6/TMPRSS2 cells were cultured at 37 °C under 5% CO₂.

Kawashiro K., Suzuki R., Nogimori T., Tsujino S., Iwahara N., Hirose T., Okada K., Yamamoto T., Fukuhara T., Hotta K, & Shinohara N. (2024). Neutralizing antibody responses and cellular responses against SARS-CoV-2 Omicron subvariants after mRNA SARS-CoV-2 vaccination in kidney transplant recipients. Scientific Reports, 14, 12176.

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Cultivation of MDCK and VeroE6/TMPRSS2 Cells

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Madin-Darby canine kidney (MDCK) cells were grown in Eagle’s minimal essential medium (E-MEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml). VeroE6 cells stably expressing transmembrane protease serine 2 (VeroE6/TMPRSS2; JCRB Cell Bank 1819) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) low glucose (catalog [cat] number 08456-65; Nacalai Tesque) supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), and G418 (1 mg/ml) (39 (link)).

Nagai M., Moriyama M, & Ichinohe T. (2021). Oral Bacteria Combined with an Intranasal Vaccine Protect from Influenza A Virus and SARS-CoV-2 Infection. mBio, 12(4), e01598-21.

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