5 Cell Culture Rules

IMPORTANT RULES FOR SUCCESSFUL CULTURING

Please read and carefully follow the specific cell culture instructions shipped with your cryovial, as well as the general guidelines listed below.

Before starting, ensure that all required reagents and supplies are prepared and readily available. Several steps require immediate, sequential action; full attention is necessary until the cells are safely seeded and placed in the incubator.

1. Proper Shipping and Storage

• To ensure maximum cell viability, cryovials must remain covered with dry ice during transit and be stored correctly upon receipt.
• Total transit time from Cell Applications to the end-user should not exceed 3–4 days. Distributors must replenish dry ice and ship to the end-user immediately upon receiving the package.
• Store cryovials in the vapor phase of liquid nitrogen if not used right away. Do not store at -80°C or in dry ice long-term.
• When removing cryovials from liquid nitrogen, bury them immediately in dry ice and initiate the thawing protocol as soon as possible..

2. Do Not Over-Thaw the Cryovials

• Thawing Time: Thaw cryovials in a 37°C water bath for no longer than 90 seconds.
• The Golden Rule: Remove the vial from the water bath while visible ice crystals still remain.
• Seeding Adherent Cells: Gently pipet to mix, then promptly transfer the cell suspension into the recommended culture flask containing the appropriate volume of pre-warmed culture medium (e.g., 15 mL for a T-75 flask).
• Seeding Suspension Cells: Gently mix and immediately dilute the cell suspension at least 1:10 with pre-warmed culture medium. Centrifuge at 400xg for 5 minutes, remove and discard the supernatant and seed the cells in culture medium.

Important: While DMSO protects cells during freezing, it is highly toxic to cells upon thawing.
Prolonged exposure to DMSO at room temperature or 37°C will significantly reduce cell viability and recovery.

3. Avoid Centrifugation Immediately After Thawing (For Adherent Cells)

Unless specifically instructed by the protocol, do not centrifuge freshly thawed adherent cells.
*Freshly thawed primary cells are extremely fragile and highly sensitive to mechanical stress.

• Seed thawed adherent cells to the flask with pre-warmed culture medium overnight before changing the media to remove DMSO.

4. Do Not Over-Trypsinize During Subculture

• Reagent Temperature: Use Trypsin/EDTA at the recommended concentration. Do not warm Trypsin/EDTA to 37°C; use it at room temperature.
• Monitoring: Monitor cells closely under the microscope during trypsinization. As soon as the cells become rounded, tap the side of the flask to dislodge them.
• Prevent Damage: Do not wait for the cells to detach spontaneously. Prolonged trypsin exposure damages cell membranes and drastically reduces viability.

5. Use Appropriate Cell Culture Ware

• Use only tissue culture-treated (TC-treated) flasks and dishes specifically designed for anchorage-dependent cell culture applications.