Expicho Expression System Protocol Guide

Component

Quantity

Cat. No.

Storage

ExpiCHO-S Cells (1 x 10⁷ cells/mL)

2 x 1 mL

A29127

Liquid nitrogen*

ExpiCHO Expression Medium

1 L

A2910001

2°C to 8°C (protect from light)

ExpiFectamine CHO Transfection Kit for

1 L of culture:

ExpiFectamine CHO Reagent

ExpiCHO Feed

ExpiFectamine CHO Enhancer

1 kit

3 x 1.1 mL

3 x 110 mL

6 mL

A29129

2°C to 8°C

2°C to 8°C (protect from light)

2°C to 8°C

OptiPRO SFM (complexation medium)

100 mL

12309050

2°C to 8°C (protect from light)

Antibody-Expressing Positive Control Vector

(at 1 mg/mL in TE buffer, pH 8)**

150 µg

A14662

–20°C

Biosafety cabinet with HEPA filter

Incubator with temperature, humidity, and CO₂ regulators

Water or metal bead bath

Centrifuge (>3000 x g)

Vortex mixer

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 medium, PBS, and sterile H₂O

70% ethanol

10% bleach

Personal protective equipment (PPE) (laboratory coat, safety goggles, disposable gloves, eye wash station)

General cell handling

• Important: All solutions and equipment that come into contact with the cells must be sterile. Always use proper sterile technique and work in a laminar flow hood or biosafety cabinet (BSC).

• Important: Store the frozen cells in liquid nitrogen until ready to use. Do not store the cells at –80°C.

• Avoid short-term, extreme temperature changes. When storing cells in liquid nitrogen, allow the cells to remain in liquid nitrogen for 3–4 days prior to the first thaw.

• For all cell manipulations, mix the cells with gentle swirling. Avoid vigorous shaking and pipetting.

• Before starting your experiments, be sure to have cells established and have some frozen stocks on hand.
• Upon receipt, grow and freeze multiple vials of ExpiCHO-S Cells to ensure that you have an adequate supply of early passage cells.

• Allow freshly thawed cells to recover in culture for two or more passages post-thaw prior to transfection.

• ExpiCHO-S Cells are adapted to high-density growth conditions with a doubling time of approximately 17 hours. They have a broad log phase growth window spanning from approximately 4 x 10⁶ to 1.5 x 10⁷ cells/mL with a maximum density of approximately 2 x 10⁷ cells/mL in shake flask cultures.

• For general maintenance, passage ExpiCHO-S Cells when they reach a density of approximately 4 x 10⁶ to 6 x 10⁶ viable cells per mL in early log phase growth, typically every 3–4 days.
• Note: Cells that are subcultured at densities outside of this early log phase growth window may have longer doubling times with lower titers over time. Modify the initial seeding density to attain the target cell density of 4 x 10⁶ to 6 x 10⁶ viable cells per mL at the time of subculturing.

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

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

Medium

• ExpiCHO Expression Medium is formulated with Gibco GlutaMAX-I Supplement. Therefore, ExpiCHO Expression Medium does not require supplementation for suspension growth and transfection applications.

Important: ExpiCHO Expression Medium is sensitive to light. For optimal results, keep it protected from light during use and storage.

Thaw cells (Day 1)

1. 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.

2. 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)

3. Carefully pipette the entire contents of the vial into the 125 mL Erlenmeyer shake flask containing 30 mL pre‑warmed ExpiCHO Expression Medium.

Incubate cells

4. 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)

5. On Day 3 post-thaw, determine viable cell density and percent cell viability. Cell viability should be ≥90%.

6. Continue to monitor cell density and viability until the culture has reached 4 x 10⁶ to 6 x 10⁶ viable cells per mL, typically three or four days post-thaw, then proceed to subculturing (next segment).

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 ExpiCHO 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 4 x 10⁶ to 6 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 4 x 10⁶ to 6 x 10⁶ viable cells per mL at the subculturing stage.

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