Expi293 Expression System Protocol Guide

Biosafety cabinet with HEPA filter

Incubator(opens in a new tab)/shaker (temperature, humidity and CO₂ regulated) 

Water or metal bead bath

Centrifuge (>3000 x g) 

Vortex

Refrigerator and freezer 

Automated cell counter

Liquid nitrogen freezer 

Aspiration pump (peristaltic or vacuum) 

Waste containers and vacuum traps

Inverted microscope

Water purification system

Cell culture flasks, vented, non-baffled (125 mL to 5 L) 

Serological pipettes and pipettors (1 mL to 50 mL) 

Multichannel pipettors and pipettes (1 µL to 200 µL) 

Single channel pipettors and pipettes (1 µL to 1 mL) 

Conical tubes 

Cryovials 

Permanent markers and labels

DMSO  

Cell culture media, PBS and sterile H₂O 

70% ethanol 

10% bleach 

Personal protective equipment (Lab coats, safety goggles, disposable gloves, eye wash station)

General cell handling

• All solutions and equipment that come in contact with the cells must be sterile. Always use proper aseptic technique and work in a laminar flow hood.

• For all cell manipulations, mix the cells by gentle swirling and avoid vigorous shaking and pipetting. Cell health is critical for maximal performance.

• Expi293F cell lines are robust cell lines adapted to high-density growth conditions with a doubling time of approximately 24 hours during log phase growth.

Guidelines for thawing and storing cells

• On receipt, either thaw the cells immediately into pre-warmed Expi293 Expression Medium or immediately place the frozen cells into vapor phase liquid nitrogen storage until ready to use. Do not store the cells at –80°C.

• Avoid short-term, extreme temperature changes. When storing cells in liquid nitrogen following receipt on dry ice, allow the cells to remain in liquid nitrogen for 3–4 days before thaw.

• Before starting experiments, ensure to have cells that are established and have frozen stocks on hand. On receipt, grow and freeze multiple vials of Expi293F cells to ensure that you have an adequate supply of early-passage cells.

Guidelines for cell maintenance and subculturing

• Allow freshly thawed cells to recover in culture for three or more passages post-thaw before transfecting.

• Use an automated cell counter or a hemocytometer with the trypan blue exclusion method to determine cell viability. Log phase cultures should be ≥95% viable.

• When thawing or subculturing cells, transfer cells into pre-warmed medium.

• Cell viability should be ≥90% within 4–7 days post-thaw with viable cell density typically >1 × 10⁶ viable cells/mL at this time; if viability is not ≥90%, cells should be incubated for up to an additional 3 days in order to reach this criterion.

• At the time of first subculture, cells should be subcultured when the viable cell density reaches 1–3 × 10⁶ viable cells/mL.

• Note: Cells that are subcultured at densities outside of this early log-phase growth window can show longer doubling times and lower protein titers over time. Modify the initial seeding density to attain the target cell density of 3–5 × 10⁶ viable cells/mL at the time of subculturing.

Guidelines for inducible cells

• For routine culture maintenance, add blasticidin to a final concentration of 20 µg/mL to culture medium.

• Blasticidin can be present in the media during cryopreservation without impacting cell health.

• An inducible expression vector must be utilized in conjunction with the Expi293F Inducible Cells and Expi293F Inducible GnTI- Cells. pcDNA 5/TO expression vector is recommended for lowest levels of basal expression and highest levels of expression upon induction with tetracycline.

• We recommend making a 1 mg/mL tetracycline stock solution in water.

Guidelines for media

• Expi293 Expression Medium is formulated with GlutaMAX Supplement. For suspension growth and transfection applications, use the Expi293 Expression Medium without any supplementation.

Thaw cells (Day 1)

1. Add 30 mL of pre-warmed Expi293 expression medium to a 125‑mL Erlenmeyer shaker flask

2. Remove the vial of cells from liquid nitrogen and swirl it in a 37°C water bath for 1–2 min to rapidly thaw the cells until only a small amount of ice remains. Note: Do not fully submerge the vial in the water bath.

3. Decontaminate the external surface of the vial by wiping it with 70% ethanol before opening the vial in the BSC or laminar flow hood.

Add cells to medium (Day 1)

4. Carefully pipette the entire contents of the vial into the 125 mL Erlenmeyer shake flask containing 30 mL pre‑warmed Expi293 expression medium.

Incubate cells

5. Incubate the cells at 37°C on the orbital shaker with ≥80% relative humidity and 8% CO₂.

Shaking speed

19 mm shaking diameter: 125 ± 5 rpm

25 mm shaking diameter: 120 ± 5 rpm

50 mm shaking diameter: 95 ± 5 rpm

Count cells and determine viability (Day 3)

6. On Day 3 post-thaw, determine viable cell density and percent cell viability. Cell viability should be ≥90% with a viable cell density >1 × 10⁶ viable cells/mL.

7. For subsequent routine cell culture maintenance, subculture cells every 3 to 4 days when the viable cell density reaches 3 × 10⁶ to 5 × 10⁶ viable cells/mL.

Passage cells (Day 1)

1. Based on the measured viable cell density, calculate the volume of cell suspension required to seed a new shake flask according to the recommended seeding densities in Table 1 and the recommended culture volumes in Table 2.

2. Transfer the calculated volume of cells to pre-warmed Expi293 Expression Medium in the Erlenmeyer shake flask(s).

Subculturing and harvest

3. Incubate flask(s) on the orbital shaker in the 37°C incubator with ≥80% relative humidity and 8% CO₂ until culture(s) reach a density of 3 x 10⁶ to 5 x 10⁶ viable cells per mL.

Note: Cells that are subcultured at densities outside of this early log phase growth window may show longer doubling times and lower titers over time. If necessary, modify the initial seeding density to attain the target cell density of 3 x 10⁶ to 5 x 10⁶ viable cells per mL at the subculturing stage.

4. Repeat Steps 1–3 to maintain or expand the cells for transfection.