How to Remove Dead Cells from Suspension Culture

If you’re working with suspension culture for the first time, you might be wondering how to remove dead cells from suspension culture. You’re not alone. While it can be relatively easy to differentiate live cells from dead ones in adherent culture, with suspension culture you have different issues to address. Thus, it is critical to understand what you’re working with before moving on to the separation and removal.

Adherent Culture vs Suspension Culture

For everything from cultivating and harvesting yeast to creating vaccines, humans have been working cell cultures. We provide a growth medium, and we can get almost any colony forming unit to grow and respond to various interventions.

Adherent cell culture was our first practice in studying cells, in which biologists place a sample of cells on a fixed growth medium, typically on a petri dish with agar, and then can watch what happens under a microscope. The process is simple and easy, and, when managed well, has provided us with answers to all kinds of questions about viruses and bacteria and allowed us to produce life-saving medicine.

Suspension culture was not far behind adherent culture, first explored in 1885 by Willhelm Roux, who placed chicken embryos in a saline buffer for several days. From there, the possibilities have only grown. Suspension culture requires a vessel filled with growth medium in which you can implant a cell culture for growth and observation. The only problem with suspension culture is that the vessel must be agitated regularly to circulate oxygen throughout the growth medium. Furthermore, cell cultures become less productive over time, so observing large quantities of growth is not always helpful. Finally, the cells may become damaged by shear conditions.

Still, while suspension culture has yet to prove itself useful in situations of vaccine creation, there are still many advantages, and the more we work with this medium, the more we can expect to learn from, develop, and improve upon it.

The Advantage of Suspension Culture

The largest advantage of suspension culture is that it allows biologists to scale up production of the cell culture. With adherent culture, cells are limited by the space on the petri dish. Ultimately, and rather quickly, the cells will need to grow beyond that space or die. In suspension culture, cells are only limited by the size of the vessel and the liquid medium within it.

With this advantage, we can watch plant, mammalian, and other living organism cells grow beyond what they are capable of with adherent culture. The trick, at that point, is to differentiate live cells from the dead ones and then remove them.

Why Remove the Dead Cells from Suspension Culture

It is important as we study colony forming units to understand how they are growing, what is inhibiting them, how many of them are dying, and why.

One way we do this is by separating the live cells from the dead ones.

Also read: How to Separate Dead Cells from Live Cell in Suspension  

In adherent culture, we can use the dye test, noting that the cells that absorb the dye are dead because they do not have the ability to “digest” and transmute the die in the way that living cells do.

With suspension culture, we typically take a different approach.

Dead Cell Removal Protocol

The standard method for identifying and removing dead cells from suspension culture is with centrifugation.

First, note that you want to be careful not to centrifuge your cells too high as that can kill off perfectly viable living cells.

Instead, in a 50 mL centrifuge tube, layer 18 mL of your cell suspension onto 12 mL of a Ficoll-paque combination. Centrifuge your tube for 15 minutes at 400x g.

When centrifugation is complete, you will note that the dead cells collect at the bottom of the tube in pellet form. From there, it should be a simple matter to remove them.

Then, you can perform a cell count.

Image Analysis for Cell Counting In Suspension Cultures

Your best chance at performing a fast and accurate cell count for suspension cultures is by using automation via an Image Analysis Software like the one designed by Oculyze. It can be successfully used to count cells within suspension culture from just an uploaded image, completely solving the problem of human error and saving time and energy in the lab. It can also differentiate between alive and dead cells, when the sample is stained, so you can also use it for your viability assessments, which are notoriously difficult to handle manually.

Find out more about the Oculyze technology on our IAP page and if you need a custom solution for optimizing your lab work, contact us and we’ll be happy to find out the details of your specific needs and find together the best way to attend to them.

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