Culturing HEK 293f Cells¶

Created by Ravishankar Madhu¶

Last updated: 2023-07-10

Description¶

This protocol describes how to Culture HEK 293f Cells used for protein production. This protocol is not a step by step explanation on how to culture these cells, but more of a list of general guidelines to follow.

Reagents and Materials¶

Incubator with a Shaker (Upper Left Incubator)
HEK 293f Cells
Freestyle Media
Vented Disposable PETG Flasks of various sizes (50mL, 125mL, 1L, 2L)

Guidelines¶

Initial Seeding¶

Initial Seeding of cells should be 5e6 of cells in 10mL of FreeStyle Media. (0.5e6 cells / mL)
This should be in a 125mL Vented Flask

Passaging the Cells¶

The cells should be passaged every 2 days
The cells should be expanded based on volume in the following order: 10mL --> 30mL --> 100mL --> 500mL --> 1L --> 2L --> 4L.
Each time the cells are passaged, they should be seeded at 0.5e6 cells / mL
A cell count should be taken every two days. If the cell count is above 3e6 / mL it is a sign that the cells should be passaged.

Harvesting the Cells for Protein¶

Transfer culture into the 500mL or 1L plastic bottles
Spin the Culture down at 4500 RPM for 25 minutes
Collect the Supernatant because that is where the proteins are

Next Steps¶

The supernatant immediately goes into RM026 - Nickle Column Protein Purification.

Appendix¶

Recipe's for Buffers¶

Freestyle Media¶

The FreeStyle Media is used to culture the HEK293f cells used in Protein Production. This media is ordered and is normally stored in 1L plastic bottles in the Cold Room. Bottles that are actively being used are stored in Eman's shelf of the Main Lab Fridge next to the Weigh Scale.

Bottles that are actively being used should be wrapped with Aluminum Foil to prevent UV-Degradation.

No additives are required before using this Buffer to Culture HEK293f cells, but the buffer should be warmed in the hot water bath before being used.