Optimum Growth® Protocol for Insect Cells

Introduction to Insect Cell Line Strains

Start-up culture from Freezer Stocks

Sterile cell culture technique must be used at all times.

In order to start a culture from a frozen stock follow the steps below:

1. Remove frozen vial of cells from liquid nitrogen vapor phase storage and quickly place in 37°C water bath.
2. Remove before fully thawed and spray outside of vial with 70% ethanol before placing it inside a biosafety cabinet.
3. Transfer cells to 125mL Optimum Growth® flask with 40mL pre-warmed media.
4. Shake flask at 135rpm (1”/25mm orbit) and 27°C
5. Take a small aliquot of the suspended cells and count them using a ViCell™ to determine density and viability.
6. Continue counting the cells on a ViCell™ every day and passage the cells when they reach the appropriate density depending on the cell line (8eX106 cells/mL for Sf9, 4e X106 cells/mL for Sf21, 3e X106cells/mL for High fives, 3eX107 cells/mL for S2).
7. Passage cells to 1e X106cells/mL once they reach a density of ~8eX106 cells/mL.

Note: The thawed cells may not look nice and round at a first glance and sometimes there might be some cell debris but this will improve with subsequent passages. It is always good practice once the cells have reached at least a third passage to freeze down several cryo-vials of cells as backup.

Freezing Insect Cells

Typically most insect cell lines can be frozen at a density of ~1 x 107 cells/ml.

1. Count the cells using ViCell™ and determine the number of freezer stocks to be made.
2. Centrifuge cells at 600xg for 5 minutes.
3. Remove the media and save
4. Prepare freezing media and place on ice for > 15 min.
5. Freezing medium: 45% conditioned (used or spent) medium, 45% fresh medium, 10% DMSO.
6. Resuspend the cells in the cold medium and aliquot 1 ml of cells into 2ml cryo-vials.
7. Put the filled vials in an isopropanol bath (Mr. Frosty) overnight.
8. Placed the freezer stocks in liquid nitrogen.

Passaging or subculturing insect cells

Steps to infect insect cell cultures for protein expression:

1. Generate a high-titer virus stock through transfection of bacmid and amplification of the virus (Expression Systems 2011)
2. Determine the titer of your virus (Expression Systems 2011).
3. Perform a small scale- expression test to determine the best multiplicity of infection (MOI) and time of infection (TOI) for expression of the target protein.
4. Passage a 2.5-3.0L of Sf9 cell culture at 1e X106cells/mL in 5L Optimum Growth® flasks the day prior to infection and shake at 135 RPM and 27°C. *This set up tends to generate the highest yield of monomeric recombinant protein at large scale.

Note: If time or resources do not permit a small scale test of a Baculovirus expression protocol, then typically an MOI of 1-5 and a TOI of 2-3 days works well.

Harvest cell cultures for future protein purification

Once the infection has been done and the culture has reached the end of the TOI:

1. Remove the Optimum Growth® flasks from the shaker.
2. Take a final cell count of the cultures on the ViCell™.
3. Pour cell cultures into centrifuge tubes and spin down at low speed (~600xg) to prevent cell lysis before downstream processing.
4. For secreted target protein:
5. Decant and save the supernatant.
6. For soluble or membrane target proteins:
7. Decant and save the cell pellet.