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Outgrown the Roost: Passaging Suspension Cells

Previously, you have learned about passaging adherent cells and read a quick protocol to make it happen. In this article, I will talk about passaging suspension cells. Some cells naturally live in suspension in body fluids and do not attach to surfaces, such as cells of hematopoietic origin found in our bloodstream.

Culturing these suspension cells is somewhat easier than adherent cell cultures because suspension cells do not require trypsinization as they are already free floating. Therefore the process of passaging is much faster and less stressful for the cells.

Basic tips for passaging suspension cells

1)   Suspension cells are usually maintained in culture flasks and reseeded when they reach confluency every 2 or 3 days.

2)   It’s not necessary to actually remove all of the old media as is done for adherent cells. Instead, a fraction of the old culture can be removed and the remaining culture can be diluted to an appropriate cell density with fresh media.

3)   Alternatively, a fraction of cells could simply be pipetted from an old flask and diluted into fresh culture media.

4)   You can tell when suspension cells reach confluency because they will begin to clump together and float on top of the medium; the medium will change color slightly and appear more turbid.

5)   Cell pelleting – if we want to collect suspension cells, an aliquot or the entire culture can be transferred into a falcon tube, or split between several falcon tubes, and centrifuged to pellet the cells.

Basics steps for passaging suspension cells

1)   View cultures under an inverted phase contrast microscope. Healthy growing suspension cells should be round and bright and there should not be a lot of cell debris. Check if the medium is acidic by looking at its color: phenol red turns yellow when the pH is acidic, indicating that you have too many cells in the culture.

2)   If the culture is acidic, centrifuge at 150 x g for 5 minutes, remove old media and reseed in fresh media. If the culture is not acidic, proceed to step 3 without centrifugation.

3)   Take about 100 ?l of the cell culture and count the cells (we’ll have an article on this soon). Calculate the cell concentration in cells per ml.

4)   Reseed the desired number of cells into freshly prepared flasks by diluting the cells with fresh medium.

5)   Repeat this every 2-3 days.

Here is a side-by-side comparison between suspension and adherent cell cultures:

Suspension cells Adherent cells
Easier to culture, can be diluted without  removing all old media More steps needed for culture, require complete media change
Do not require mechanical or chemical dissociation Require trypsinization to subculture, is stressful for the cells
Not easy to determine confluency, require daily cell count Can be easily inspected under a microscope to determine confluency
Growth limited by cell concentration Growth limited by surface area

 

 

 

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