Key Considerations for Culturing NCI-H520

The NCI-H520 cell line was established by A.F. Gazdar et al. in 1982 from a lung specimen of a patient with squamous cell carcinoma of the lung. These cells exhibit epithelial-like morphology and are adherent in nature. This guide will provide you with detailed information on the characteristics, culture procedures, and important considerations for NCI-H520 cells, helping you to efficiently set up your experiments.

Cell Information

1. Cell Name: NCI-H520 (Human Lung Squamous Cell Carcinoma)
2. Aliases:?H520; H-520; NCI-HUT-520; NCIH520
3. Growth Characteristics:?Epithelial-like, adherent, growing in an island pattern
4. Doubling Time:?32-60 hours
5. Cell Culture Conditions:

• Medium:?RPMI-1640＋10%FBS＋1%P/S
• Atmosphere: 95% air, 5% CO2
• Temperature:?37°C

6. Medium Change Frequency: 2-3 times per week

7.?Subculturing Ratio: 1:2 to 1:4 (adjust based on actual cell density)

Subculturing Procedure

1??Aspirate Medium:?Remove the spent culture medium.

2??Rinse:?Add approximately 2 mL of PBS, gently swirl to rinse the cells, and then aspirate the PBS.

3??Add Trypsin:?Add about 1 mL of 0.25% trypsin solution (with EDTA), gently swirl to ensure it covers all cells.

4??Incubate:?Place the flask in the incubator. Observe under a microscope; when cells in the center of clusters become round and distinct gaps appear (about 3-6 minutes), gently swirl the flask. When most cells detach, stop the digestion by adding 3 mL of serum-containing medium. Do not tap the flask?throughout the entire process.?Gently pipette to dislodge cells, ensuring they are suspended as single cells.

5??Centrifuge:?Collect the cell suspension and centrifuge at 1200 rpm for 3 minutes. Discard the supernatant.

6??Resuspend:?Add fresh medium, gently pipette to mix, then seed into new culture flasks at the desired ratio. Loosen the cap or use a vented cap, and return the flask to the incubator. Monitor cell growth regularly.

Medium Change Procedure

Medium Change:?Aspirate the old medium and gently add fresh medium along the side of the flask (avoid the cell attachment area). Change the medium every 2-3 days.

Important Considerations

• Island-like Growth:?These cells grow in an island pattern. It is crucial to control digestion time accurately. Too short a digestion time makes it difficult for cells to detach, and excessive pipetting can lead to increased cell fragmentation. Aggregates that do not disperse can hinder cell growth. Conversely, too long digestion may result in more floating cells post-passage.
• Microscopic Observation:?It is recommended to observe the cells under a microscope every minute during digestion. Stop digestion when cells start to separate, ensuring cells are as dispersed as possible to avoid clumping.
• Gentle Handling:?During cell passage, pipette gently to avoid creating excessive bubbles that can increase cell fragmentation.
• Floating Debris: It is normal to see some floating cell debris in the medium, which may appear as small black dots. Medium changes can temporarily reduce this, but some debris will reappear. This does not affect cell proliferation or experimental outcomes.
• Serum Concentration Adjustment:?If cell growth is slow, increasing the serum concentration by 5-10% (total serum concentration not exceeding 20%) may help. Once cells recover, revert to the normal serum concentration.
• Adhesion and Recovery:?NCI-H520 cells adhere and spread slowly. After 24 hours post-recovery, most cells will be irregular polygons or round. By 48 hours, cells will start to grow in clusters. Newly recovered cultures may have more floating cells; avoid changing the medium within the first 48 hours to allow floating cells to reattach.

NCI-H520 cells exhibit moderate growth speed. When cell confluency reaches above 80%, they are ready for passage.

Subculturing Ratios and Timing:

• 1:2 Subculture Ratio: Cells can be passaged again in approximately 2-3 days.
• 1:3 Subculture Ratio: Cells can be passaged again in approximately 3-4 days.