Cell Biology JoVE (Open Access) Citable · DOI

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

Name: Scale-Up of Mammalian Cell Culture using a New Multilayered Flask
Uploaded: 2026-08-06
Duration: 8 min 39 s
Description: A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

DOI: 10.3791/3418-v

What you'll learn

✓ Set up and prepare multilayered flasks for efficient mammalian cell culture expansion
✓ Mix and equilibrate cells across flask layers to ensure homogenous population distribution
✓ Execute media exchange and cell harvesting protocols in multilayered flask systems
✓ Optimize working volumes and scaling parameters for attachment-dependent cell production

Protocol

Biopharma Insights Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

Difficulty

intermediate

Total time

~1–2 hours per flask (culture dependent); 24–72 hours for full growth cycle

Model organism

Mammalian cell culture (attachment-dependent; specific line not specified)

Biosafety

BSL-1

Steps

Design and prepare multilayered flask with cell suspension

Assemble the Falcon Multi-Flask and prepare it for cell inoculation. Add the appropriate cell suspension volume according to manufacturer specifications and experimental requirements.

▶ 01:00

Mix cells and equilibrate fluid distribution across layers

Perform gentle mixing to ensure cells distribute evenly across all flask layers and achieve equilibration of culture media throughout the multilayered structure.

▶ 02:19

Transport and position multilayered flask for incubation

Move the prepared flask to the incubation environment using appropriate handling procedures. Ensure proper placement for consistent gas exchange and temperature control.

▶ 03:23

Exchange culture media and harvest cells from flask

Perform routine media changes to maintain nutrient availability and pH. Execute cell harvesting procedures suitable for multilayered flask geometry.

▶ 04:12

Determine optimal working volume for flask layers

Identify and apply recommended working volumes to maximize cell yield while maintaining adequate gas exchange and nutrient distribution across all layers.

▶ 05:40

Verify homogenous cell population across multilayered structure

Confirm that cells have distributed uniformly across all flask layers and that the culture population remains homogenous for consistent downstream applications.

▶ 06:10

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