Expicho s

Manufactured by Thermo Fisher Scientific

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The ExpiCHO-S is a suspension-adapted Chinese Hamster Ovary (CHO) cell line designed for high-density transient transfection and rapid recombinant protein production. The cell line is optimized for use in fed-batch cultures and can achieve high viable cell densities and protein yields.

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ExpiCHO-S cells (74 citations) ExpiCHO (30 citations) ExpiCHO-S expression system (4 citations) ExpiCHO-S cell line (2 citations)

26 protocols using expicho s

Protein Production in Expi Cell Lines and Huh7.5.1-ACE2-TMPRSS2 Cell Culture

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ExpiCHO-S™ (ThermoFisher Scientific, Catalog number: A29127) and Expi293F™ (ThermoFisher Scientific, Catalog number: A14527) cells were used to produce recombinant proteins. Both cell lines were maintained according to the manufacturer recommended protocol until transfection. ExpiCHO-S™ (1ml, 1x10⁷cells) were directly thawed into 25ml of prewarmed ExpiCHO™ expression media and allowed to revive for 2-3 days. The cells were passaged every 3-4 days to maintain density at or below 4x10⁶ - 6x10⁶ viable cells/ml. ExpiCHO-S™ cells were maintained at 37°C, 8% CO2 and 125 +/- 5rpm. Expi293F™ (1ml, 1x10⁷cells) were directly thawed into 25ml of prewarmed Expi293™ expression media and allowed to revive for 2-3 days. The cells were passaged every 3-4 days to maintain density at or below 3x10⁶ - 5x10⁶ viable cells/ml. Expi293F™ cells were maintained at 37°C, 8% CO2 and 125 +/- 5rpm
Huh7.5.1-ACE2-TMPRSS2 cell line was a gift from Andreas Puschnik. 1x10⁷- 1.5x10⁷ cells were thawed directly into prewarmed complete culture media (Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum, 10mM HEPES, 1 × Pen-Strep-Glutamine). The cells were allowed to revive for 2-3 days and were passaged once they reached >70-80% confluence and were maintained at >95% viability in complete culture media. Huh7.5.1-ACE2-TMPRSS2 cells were maintained at 37°C and 5% CO2.

Remesh S.G., Merz G.E., Brilot A.F., Chio U.S., Rizo A.N., Pospiech TH J.r., Lui I., Laurie M.T., Glasgow J., Le C.Q., Zhang Y., Diwanji D., Hernandez E., Lopez J., Mehmood H., Pawar K.I., Pourmal S., Smith A.M., Zhou F., DeRisi J., Kortemme T., Rosenberg O.S., Glasgow A., Leung K.K., Wells J.A, & Verba K.A. (2023). Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps. Structure (London, England : 1993), 31(3), 253-264.e6.

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Vero E6 and ExpiCHO-S Cell Culture

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Vero E6 (ATCC® CRL-1586^TM) were obtained from the American Type Culture Collection (Summit Pharmaceuticals International, Japan). Cells were used and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), MEM non-essential amino acids, 2 nM L-Glutamine, 100 Units/ml Penicilin, 0.1 mg/ml streptomycin and 12.5 Units/ml Nystatin (Biological Industries, Israel). Cells were cultured at 37 °C, 5% CO₂ at 95% air atmosphere.
ExpiCHO-S (Thermoscientific, USA, Cat# A29127) were used for expression of recombinant proteins as described above.

Noy-Porat T., Makdasi E., Alcalay R., Mechaly A., Levy Y., Bercovich-Kinori A., Zauberman A., Tamir H., Yahalom-Ronen Y., Israeli M., Epstein E., Achdout H., Melamed S., Chitlaru T., Weiss S., Peretz E., Rosen O., Paran N., Yitzhaki S., Shapira S.C., Israely T., Mazor O, & Rosenfeld R. (2020). A panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes. Nature Communications, 11, 4303.

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Mammalian Expression and Purification of Monoclonal Antibodies

Cited 3 times

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Mammalian expression plasmids [41 (link)] encoding the heavy and light chains of mAbs 4G2 [42 (link)], C8 [24 (link)], 513 [43 (link)], and DV62.5 [22 (link)] were transfected into ExpiCHO-S (ThermoFisher Scientific, Waltham, MA, USA) cells as per the manufacturer’s instructions and were incubated on an orbital shaker at 120 rpm with 7.5% CO₂ for seven days at 37 °C. The supernatant was harvested from the cells via centrifugation at 4800× g for 30 min, filtered through 0.22 μm filters (Merck Millipore, Burlington, MA, USA), and purified using AKTA-FPLC affinity chromatography with a 1 mL HiTrap Protein A HP column (GE Healthcare, Chicago, IL, USA). Eluate was buffer exchanged into PBS and was concentrated using 30 kDa molecular weight cut-off centrifugal filter units (Merck Amicon, Burlington, MA, USA).

Scott C.A., Amarilla A.A., Bibby S., Newton N.D., Hall R.A., Hobson-Peters J., Muller D.A., Chappell K.J., Young P.R., Modhiran N, & Watterson D. (2021). Implications of Dengue Virus Maturation on Vaccine Induced Humoral Immunity in Mice. Viruses, 13(9), 1843.

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Antibody Production for CAR-T Targeting CD19

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An antibody against the variable region of the αCD19 FMC63 present on the CAR was produced by transfection of ExpiCHO-S (Thermo Fisher Scientific) using plasmids coding for the heavy chain anti-FMC63-muIgG2a and the light chain anti-FMC63-muIgk (clone 136.20.1). ÄKTA start was used for protein purification with a HiTrap Protein G HP column according to the manufacturer instructions (GE HealthCare). The protein was dialysed with Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific) in 1000× PBS at 4°C overnight. Quantification of protein was performed using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Lopez E., Karattil R., Nannini F., Weng-Kit Cheung G., Denzler L., Galvez-Cancino F., Quezada S, & Pule M.A. (2023). Inhibition of lactate transport by MCT-1 blockade improves chimeric antigen receptor T-cell therapy against B-cell malignancies. Journal for Immunotherapy of Cancer, 11(6), e006287.

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Mammalian cell-based recombinant protein expression

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Chinese Hamster ovary cell line ExpiCHO-S™ (Thermo Fisher) and Human embryonic kidney fibroblast cell line Expi293F™ (Thermo Fisher) were used for mammalian expression of recombinant proteins for affinity measurements, crystallography and cryo-EM. Cells were maintained and transfected according to protocols in the ExpiCHO expression system™ kit (Thermo Fisher #A29133) and the Expi293 expression system™ kit (Thermo Fisher #A14635) respectively, with the addition of lupin peptone (Cell Biosciences #A230100) 24 hours post-transfection to Expi293F cells.

Wheatley A.K., Pymm P., Esterbauer R., Dietrich M.H., Lee W.S., Drew D., Kelly H.G., Chan L.J., Mordant F.L., Black K.A., Adair A., Tan H.X., Juno J.A., Wragg K.M., Amarasena T., Lopez E., Selva K.J., Haycroft E.R., Cooney J.P., Venugopal H., Tan L.L., O Neill M.T., Allison C.C., Cromer D., Davenport M.P., Bowen R.A., Chung A.W., Pellegrini M., Liddament M.T., Glukhova A., Subbarao K., Kent S.J, & Tham W.H. (2021). Landscape of human antibody recognition of the SARS-CoV-2 receptor binding domain. Cell Reports, 37(2), 109822.

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Cell Culture Conditions for Expi293-F and ExpiCHO-S

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Expi293-F and ExpiCHO-S cells were obtained from Thermo Fisher Scientific. Cells were incubated at 37 °C, 8% CO₂, 110 rpm, and sub-passaged every 3–4 days in their respective expression media, as described in the manufacturer’s protocol (Thermo Fisher Scientific, Schwerte, Germany). Cell count and viability were measured using an automated cell counter (Bio-Rad TC-20) based on trypan blue staining. Cell densities were maintained between 0.3–4 × 10⁶ cells/mL and 0.2–6 × 10⁶ cells/mL for Expi293-F and ExpiCHO-S, respectively.

Carrara S.C., Fiebig D., Bogen J.P., Grzeschik J., Hock B, & Kolmar H. (2021). Recombinant Antibody Production Using a Dual-Promoter Single Plasmid System. Antibodies, 10(2), 18.

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Cell Culture Conditions for SARS-CoV-2 Research

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Vero E6, CaCO2, A549, ACE2.A549, BHK, Huh7 293T, Vero and CHME3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S) at 37°C in 5% CO₂. CaCO2 cells were cultured in DMEM/10% FBS/1% nonessential amino acids. U937 cells were cultured in RPMI/10% FBS/ with P/S. ExpiCHO-S (Thermo Fisher Scientific) were cultured in ExpiCHO expression medium at 37 °C in 8% CO₂. Cell line ACE2 expression levels were quantified by staining with anti-ACE2 antibody (NOVUS) and Alexa Fluor 594-conjugated goat anti-mouse IgG (Biolegend) and pacific blue viability dye. Data were analyzed by flow cytometry with Flowjo software. ACE2.293T cells were established by lipofection of 293T cells with pLenti.ACE2-HA using lipofectamine 2000 (Invitrogen). After 2 days, the cells were selected in 1 μg/ml puromycin and cloned at limiting dilution. Single cell clones were expanded and analyzed by flow cytometry and a single clone was chosen. A549 cells were transfected with pLenti.ACE2 using lipofectamine 2000. After two days, the cells were selected in medium containing 1 μg/ml puromycin and cloned at limiting dilution. Individual cell clones were screened by flow cytometry for high ACE2 expression and a single cell clone was expanded.

Tada T., Fan C., Chen J.S., Kaur R., Stapleford K.A., Gristick H., Dcosta B.M., Wilen C.B., Nimigean C.M, & Landau N.R. (2020). An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2. Cell Reports, 33(12), 108528.

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Vero E6 and ExpiCHO-S Cell Culture

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Vero E6 (ATCC® CRL-1586^TM) were obtained from the American Type Culture Collection. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), MEM non-essential amino acids (NEAA), 2 mM l-glutamine, 100 Units/ml penicillin, 0.1 mg/ml streptomycin and 12.5 Units/ml Nystatin (P/S/N) (Biological Industries, Israel). Cells were cultured at 37 °C, 5% CO₂ at 95% air atmosphere.
ExpiCHO-S (Thermoscientific, USA, Cat# A29127) were used for expression of recombinant proteins as described above.

Rosenfeld R., Noy-Porat T., Mechaly A., Makdasi E., Levy Y., Alcalay R., Falach R., Aftalion M., Epstein E., Gur D., Chitlaru T., Vitner E.B., Melamed S., Politi B., Zauberman A., Lazar S., Beth-Din A., Evgy Y., Yitzhaki S., Shapira S.C., Israely T, & Mazor O. (2021). Post-exposure protection of SARS-CoV-2 lethal infected K18-hACE2 transgenic mice by neutralizing human monoclonal antibody. Nature Communications, 12, 944.

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Recombinant Chimeric Antibody Expression in ExpiCHO-S Cells

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ExpiCHO-S (Thermo scientific, A29127) cells were used for expression of recombinant chimeric full-length Abs as detailed below/above.

Rosenfeld R., Alcalay R., Zvi A., Ben-David A., Noy-Porat T., Chitlaru T., Epstein E., Israeli O., Lazar S., Caspi N., Barnea A., Dor E., Chomsky I., Pitel S., Makdasi E., Zichel R, & Mazor O. (2022). Centaur antibodies: Engineered chimeric equine-human recombinant antibodies. Frontiers in Immunology, 13, 942317.

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Murine MAb 10H4 Production and Characterization

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Murine MAb 10H4 (CPV-2 VP2 specific) was obtained from the National Research Center for Veterinary Medicine, which was produced by the immunization of mice with partially purified new CPV-2a virus (strain CVCC AV298). The host cell lines used in the expression of recombinant antibodies included the Expi CHO-S (Thermo Scientific, USA) and CHO-S cell lines (Thermo Scientific, USA). The virus of new CPV-2a, new CPV-2b, and CPV-2c were provided by the National Research Center for Veterinary Medicine.

Zhou L., Wu H., Du M., Song H., Huo N., Chen X., Su X., Li W., Wang L., Wang J., Huang B., Tan F, & Tian K. (2022). A canine-derived chimeric antibody with high neutralizing activity against canine parvovirus-2. AMB Express, 12, 76.

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