Thursday, September 4, 2008

Are We Missing Something?

Long ago when I first started working with mammalian cell cultures performing cell counts of bioreactor samples was a bit of a tedious process. You had to stain the sample with Trypan blue, load the hemacytometer slide and place it under the microscope and then proceed to tally the number of live and dead cells contained within a certain number of squares on a grid pattern that was etched onto the slide. One important element of this procedure was to make sure and choose the correct dilution factor for the sample so that you wouldn't load a sample on the slide that had so many cells that it was unreasonable to count. Hopefully, if your cell culture process is doing well, the number of cells in the bioreactor are increasing each day and you would therefore need to increase the dilution factor of your sample to account for this. (Typically the cell density in the reactor plateaus at a point in the culture and further increases in dilution factor become unnecessary.) In our group, we would typically perform two cell counts for each bioreactor sample and average the results to obtain the total and viable cell density for the culture. This process wasn't so bad when we had only a few bioreactors in the lab but over time more reactors were added and the burden of counting cells increased. At its most challenging, it could take a person between 1-2 hours to count samples from 8 bench scale bioreactors. It could be painful!

Back in 1999, Innovatis, a German company, developed an automated cell counting instrument known as Cedex that is based on the same trypan blue dye exclusion method that is traditionally used to count mammalian cells. When I first saw the Cedex in mid-2000 I was a bit skeptical that it could really do a good job counting cells but after working with the instrument for a bit and after conducting a head-to-head comparison between it and a hemacytometer, it became clear that this was a new, more efficient way, of counting cells. Because it was an automated instrument this meant that all cell counts would be handled in exactly the same manner; no more concerns about dilution errors during sample preparation; no more concerns that an operator becomes less efficient on their 15th cell count compared to their first cell count. Additionally the instrument was capable of counting a greater number of cells per sample and could could also provide information on cell size distribution. With the Cedex a cell count took only about 2-3 minutes per sample including cleaning. This meant we would count samples from 8 bioreactors in about 30 minutes!

There are now additional automated cell counters available. Beckman Coulter has developed the Vi-Cell series of counters and Nova Biomedical has their BioProfile Flex which is an automated modular analyzer for cell culture analysis that has a cell density / cell viability module.

Having these automated methods of cell counting has certainly helped streamline the process of monitoring cell cultures and improved the ability of scientists and engineers to develop improved processes. The only minor downside to the advent of these remarkable instruments is that people who are new to the bioprocess industry may never have to learn how to perform cell counts by hand.

When using a microscope and a hemacytometer to count cells we would also have the opportunity to look at the condition of the cells over the duration of a culture and observe changes in cell morphology. These observations, along with the cell count data, would provide overall "picture" of how the cells in a culture are doing. Although I would not give up the benefits that automated cell counters have added to bioprocess development, are we going to forget to look at the cells under a microscope anymore?

2 comments:

Jean said...

Great post! Why is it a downside if you don't know how to count cells by hand? Seems obsolete to me.

Anonymous said...

if your reagent pack is empty and your lab tech forgot to order a new one, a manual count comes in handy...