Vp sfm

Vero cells were adapted to grow in serum-free medium VP-SFM (ThermoFisher Scientific) by increasing the VP-SFM content in four steps (25, 50, 75, and 100%). Vero cells growing in serum-free medium therefore had a higher passage number (P174) than the cells in serum-containing medium. The cells were detached and re-suspended using the procedure described above for cells in serum-containing medium, except the trypsinization step lasted only 5 min and was stopped by adding 0.02 mL cm⁻² trypsin inhibitor (Sigma-Aldrich).

Cellastim S, recombinant human serum albumin derived from a nonmammalian host and Optiferrin, recombinant human transferrin derived from a nonmammalian host (InVitria, Junction City, KS) were reconstituted according to the manufacturer's instructions. Sterile‐filtered liquid stocks were maintained at 4°C for 1 month. EMEM (MediaTech, Manassas, VA) supplemented with FBS (ATCC, Manassas, VA) was used as the traditional FBS‐containing medium control in cell expansion and virus culture experiments. VP‐SFM (Thermo Fisher Scientific, Waltham, MA) was supplemented with 4 mM Glutamine (Thermo Fisher Scientific) and is referred as VP‐SFM throughout the text.

Madin-Darby Canine Kidney (MDCK) cells were cultured in Eagle’s Minimum Essential Medium (EMEM, Sartorius Stedim BioWhittaker) supplemented with 10% fetal bovine serum (FBS, Greiner Bio-One), 20 mM HEPES (Lonza BioWhittaker), 0.1% CHNaO₃ (Lonza BioWhittaker), and 100 μg/ml penicillin, 100U/ml streptomycin and 2mM L-Glutamine (P/S/G, Lonza). CEF were isolated from 11-day-old chicken embryos (Drost Loosdrecht BV) and passaged once before use as described previously^{45 (link)}. CEF were cultured in Virus Production-Serum Free Medium (VP-SFM, Gibco) containing P/S. HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Lonza) supplemented with 10% FBS, 20 mM HEPES, 0.1% CHNaO₃ and P/S/G. Baby Hamster Kidney (BHK)-21 cells were cultured in DMEM supplemented with 10% FBS, 20 mM HEPES, 0.1% CHNaO₃, 0.1 mM non-essential amino acids (NEAA, Lonza) and P/S/G. HeLa cells were cultured in DMEM supplemented with 10% FBS, 20 mM HEPES and 0.1% CHNaO₃ and P/S/G. All cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂.

CEFs were isolated from 11-day-old chicken embryos (Drost Loosdrecht BV) and passaged once before use. CEFs were cultured in virus production serum-free medium (VP-SFM, Gibco) containing penicillin and streptomycin. Baby hamster kidney 21 (BHK-21, American Type Culture Collection (ATCC)) cells were cultured in DMEM (Lonza) supplemented with 10% FBS, 20 mM HEPES, 0.1% CHNaO₃, 0.1 mM non-essential amino acids (Lonza) and penicillin and streptomycin/l-glutamine. HeLa cells (ATCC) were cultured in DMEM supplemented with 10% FBS, 20 mM HEPES, 0.1% CHNaO₃ and penicillin and streptomycin/l-glutamine. Vero cells (ATCC) were cultured in DMEM (Capricorn Scientific) supplemented with 10% FBS, 20 mM HEPES and penicillin and streptomycin/l-glutamine. Calu-3 cells (ATCC) were cultured in Opti-MEM + GlutaMAX (Gibco) supplemented with 10% FBS. All cell lines were grown at 37 °C in a humidified CO₂ incubator.

Vero cells were seeded and cultured in 24-well plates using serum-free medium (VP-SFM) (Gibco, Carlsbad, CA) containing 4 mM glutamine at 37°C with 5% CO₂, and the cells were grown to confluence. Varying amounts of TcdA rRBD were added to the wells, and PBS was used as the negative control. After a 30-min incubation, the wells were washed twice with cold PBS. Radioimmunoprecipitation assay buffer (RIPA buffer) (Millipore, Billerica, MA), a strong cell lysis buffer, was added to the cells. The total cell lysate was analyzed by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Pall Corporation) pre-soaked in methanol. PVDF membranes were blocked with 5% (w/v) nonfat dry milk in PBS and sequentially exposed to 0.5 μg/mL TcdA-specific monoclonal antibodies (Clone PCG-4; GeneTex, Taiwan) and 50 ng/mL horseradish peroxidase (HRP) conjugated to a secondary antibody (GeneTex) to detect the amount of TcdA rRBD bound to Vero cells. Antibodies were diluted in PBS containing 1% nonfat dry milk (w/v). PVDF membranes were washed twice at every step with PBST (1× PBS containing 0.05% Tween 20) at room temperature. Finally, signals were detected using enhanced chemiluminescence (ECL) according to the manufacturer’s instructions (KPL, Gaithersburg, MD).

Four culture media free of animal components were used in this study, including VP-SFM (Gibco^®), CD-CHO (Gibco^®), CD-Hydrolyzed (Sigma-Aldrich, St. Louis, MO, USA), and CD-CHO (Sigma-Aldrich St. Louis, MO, USA). The Advanced-DMEM/F12 (Gibco^®) that contains animal components was used as the control for B. bigemina growth (Table 1).
All culture media were buffered with 25 mM 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino] ethane sulfonic, N-[Tris(hydroxymethyl)methyl]-2-aminoethanosulphonic (TES) (Sigma-Aldrich, St. Louis, MO, USA). An antioxidant mixture was added to 2 mM of L-glutamine (Sigma-Aldrich, St. Louis, MO, USA) (GIBCO^®). The pH was adjusted (6.8) for all culture media and sterilized by filtration with a 0.22 µm membrane (Millipore). All media were supplemented with different concentrations of CD-Lipid (CORNING^®) (Table 2).