VESOCHO™ MAMMALIAN CELL PROTEIN EXPRESSION kit protocol

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Culture & Passaging

• Seeding: Inoculate at 0.3 × 10^6 cells/mL; passage or expand every 3 days at 37 °C, 120 rpm (50 mm orbit), 5% CO₂.
• Pre-transfection prep (Day −1): Adjust culture to 2 × 10^6 cells/mL so that on the day of transfection the viable cell density is ~6 × 10^6 cells/mL.

Transfection (example: 125-mL shaker flask, 20 mL working volume)

Scale all additions proportionally for other volumes.

1. Harvest/refresh: Spin seed cells at 200 × g, 5 min. Discard the medium and resuspend in fresh basal medium to ~12 × 10^6 cells/mL.
2. Add DNA: Add 80 µg plasmid DNA (4 µg/mL final). Gently swirl to mix.
3. Add Booster: Add 60 µL VESOCHO™ Booster (3 µL/mL final). Mix gently.
4. Add Transfection reagent: Slowly add 150 µL VESOCHO™ Trans (7.5 µL/mL final) while swirling to mix thoroughly.
5. Incubate (mark as Day 0): Place on shaker at 37 °C, 120 rpm (50 mm orbit), 5% CO₂.

Feeding strategy

Day 1 (24 h post-transfection)

Add the following to each culture:

• VESOCHO™ Enhancer: 60 µL (0.3% v/v) — single addition only
• VESOCHO™ Feed: 1.8 mL (9% v/v)
• Glucose: Adjust to 6 g/L
• Temperature shift: Reduce to 32 °C

Harvest options

• Day 7 harvest: Add 1.8 mL (9% v/v) VESOCHO™ Feed on Day 5 → harvest on Day 7.
• Day 14 harvest: Add 1.8 mL (9% v/v) VESOCHO™ Feed on Day 5 and Day 9; add glucose 8 g/L on Day 7 and Day 11 → terminate when viability < 60% or at the planned endpoint.

Notes

• The supplied control antibody plasmid is pre-mixed at HC:LC = 1:2 and can be used directly. For your own constructs, you may adjust HC/LC ratio and total DNA as needed while keeping the total DNA amount constant.
• If the shaker orbit < 50 mm, increase rpm appropriately.
• Recommended working volume is 15–20% of the nominal flask volume.

Culture & Passaging

• Seeding: Inoculate at 0.3 × 10^6 cells/mL; passage or expand every 3 days at 37 °C, 120 rpm (50 mm orbit), 5% CO₂.
• Pre-transfection (Day −1): Adjust culture to 2 × 10^6 cells/mL so that on transfection day the viable cell density is ~6 × 10^6 cells/mL.

Transfection (example: 125-mL shaker flask, 20 mL working volume)

Scale all additions proportionally for other volumes.

1. Set cell density: Using fresh basal medium, adjust the culture to ~6 × 10^6 cells/mL.
2. Add DNA: Add 60 µg plasmid DNA (3 µg/mL final). Gently swirl to mix.
3. Add Booster: Add 60 µL VESOCHO™ Booster (3 µL/mL final). Mix gently.
4. Add Transfection Reagent: Slowly add 150 µL VESOCHO™ Trans (7.5 µL/mL final) while swirling to ensure uniform mixing.
5. Incubate (mark as Day 0): Place on shaker at 37 °C, 120 rpm (50 mm orbit), 5% CO₂.

Feeding Strategy

Day 1 (24 h post-transfection)

Add to each culture:

• VESOCHO™ Enhancer: 60 µL (0.3% v/v) — single addition only
• VESOCHO™ Feed: 1.8 mL (9% v/v)
• Glucose: Adjust to 6 g/L
• Temperature shift: Reduce to 32 °C

Harvest options

• Day 7 harvest: Add 1.8 mL (9% v/v) VESOCHO™ Feed on Day 5 → harvest on Day 7.
• Day 14 harvest: Add 1.8 mL (9% v/v) VESOCHO™ Feed on Day 5 and Day 9; add glucose 4 g/L on Day 7 and 6 g/L on Day 11 → stop when viability < 60% or at the planned endpoint.

Notes

• The supplied control antibody plasmid is pre-mixed at HC:LC = 1:2 and can be used directly. For your own constructs, you may adjust the HC/LC ratio and amounts as needed while keeping the total DNA constant.
• If the shaker orbit < 50 mm, increase rpm appropriately.
• Recommended working volume is 15–20% of the nominal flask volume.

A) Direct Adaptation

• Cells cultured directly into VESOCHO™ Basal at 0.3–0.5 × 10^6 cells/mL in 125-mL shaker flasks with 20–30 mL working volume (adjust as needed to match your existing process). Culture at 37 °C, 120 rpm (50-mm orbit), 5% CO₂.
• Passage every 3 days until growth is stable. If the doubling time is no longer than in the control medium, the cells are considered adapted to VESOCHO™ Basal.

B) Gradual (Stepwise) Adaptation

Use this if the cell line does not adapt directly to VESOCHO™ Basal. Ratios below can be adjusted based on performance.

1.  When the culture in the original medium reaches viable cell density (VCD) ≥ 2 × 10^6 cells/mL and viability ≥ 90%, begin stepwise adaptation by replacing with mixtures of VESOCHO™ Basal : Original medium as shown. At each step, inoculate at 0.3–0.5 × 10^6 cells/mL, confirm good growth, then move to the next ratio.

VESOCHO™ Basal : Original (%)    Inoculation Density (×10^6 cells/mL)    Proceed When (per step)

25 : 75                                                               0.3–0.5                                                                VCD ≥ 2 × 10^6, Viability ≥ 90% 50 : 50                                                               0.3–0.5                                                                VCD ≥ 2 × 10^6, Viability ≥ 90% 75 : 25                                                               0.3–0.5                                                                VCD ≥ 2 × 10^6, Viability ≥ 90% 100 : 0                                                               0.3–0.5                                                                VCD ≥ 2 × 10^6, Viability ≥ 90%

Note: Depending on how easily the line adapts, you may skip certain ratios or insert intermediate ratios.

2.  After 3 days in 100% VESOCHO™ Basal, the viable cell density should be ≥ 2 × 10^6 cells/mL with viability ≥ 90%. At this point, the cells are considered fully adapted to VESOCHO™ Basal.

1. Based on the target final culture volume for your project, calculate the required seed and fresh medium so that the inoculation density is 0.3 × 10^6 cells/mL.
2. In a biosafety cabinet, transfer the required seed volume into a shaker flask containing pre-warmed (37 °C) VESOCHO™ Basal.
3. Culture at 37 °C, 120 rpm (50-mm orbit), 5% CO₂.
4. Passage every 3 days using fresh medium, following the steps above.

1. Select mid-log phase cells with viability ≥ 90%.
2. Determine the viable cell density and calculate the seed volume needed; recommended freezing density: 1–2 × 10^7 cells/mL.
3. Prepare freezing medium: 90% VESOCHO™ Basal + 10% DMSO (prepare fresh, keep cold).
4. Harvest the cell seed by centrifugation at 200 × g for 5 min; discard the supernatant.
5. In a biosafety cabinet, resuspend the pellet in cold freezing medium and aliquot into cryovials.
6. Place vials in a controlled-rate freezing container and store at −80 °C overnight; transfer to liquid nitrogen for long-term storage.

1. Pre-warm a water bath to 37.0 °C.
2. Remove a vial from liquid nitrogen and thaw rapidly in the water bath (< 3 min).
3. Transfer the entire contents to a 125-mL shaker flask containing 30 mL pre-warmed VESOCHO™ Basal.
4. Culture at 37 °C, 120 rpm (50-mm orbit), 5% CO₂.
5. Passage at least twice; once fully recovered with viability > 90%, proceed with downstream work as planned.