The U.S. Congress has also recognized the importance of cell counting and cell viability measurement standards in the 21st Century Cure Act for cell and gene therapy. On March 31, 2014, the U.S. Food and Drug Administration (FDA) hosted a workshop entitled “Synergizing Efforts in Standards Development for Cellular Therapies and Regenerative Medicine Products”. With the inclusion of the National Institute of Standards and Technology (NIST) and other stakeholders, cell counting and viability measurement assurance were identified as important standards for improving the quality of cell therapy products (4).
What is trypan blue?
Trypan blue is an azo dye (m.w. 960 Da) that has been used for cell viability measurements for over a century (5,6). It can concentrate in membrane-compromised dead or dying cells, but is excluded from membrane-impermeable live cells (7,8). Although many issues have been documented for trypan blue, such as protein aggregation (9-11), limited counting time window (12), and inaccurate measurement when viability is less than 80% (13-15), it remains the go-to viability dye.
Since 2012, we have published three articles detailing cell counting issues with trypan blue staining. The first publication demonstrated that bright field counting with trypan blue can over-count live peripheral blood mononuclear cells (16). The second publication quantitatively showed that trypan blue can transforming dead cells to dim and diffuse shapes for Jurkat cells and primary mouse splenocytes, which caused over-estimation of viability when cell viability falls below 80% (15). We have further investigated the formation of the diffuse objects in the third article that was recently published (17).
What are these dim and diffuse objects formed after trypan blue staining?
Traditionally, these diffuse objects are difficult to see in microscopy images, causing researchers to under-count dead cells and over-estimate viability. In contrast, the optical components in the Cellometer instruments are able to clearly image the dim and diffuse objects in Jurkat, mouse splenocytes, and human PBMCs.
In this work, we recorded the interaction between trypan blue and cells in real-time to prove the rupturing of cells changing to the diffuse objects. With the use of fluorescent viability dye (propidium iodide, PI) that stained the nuclei of membrane-compromised dead cells, we demonstrated that these diffuse objects contained a nucleus.
Finally, we hypothesized that increase in osmotic pressure led to the rupturing of the dead or dying immune cells. This hypothesis was tested by changing the buffer concentrations and quantifying the size and color change of the trypan blue stained cells.
The visual and quantitation evidence recorded in these experiments revealed that trypan blue can induce the formation of these diffuse objects and ultimately leads to over-estimation of viability, which may have significant effects for downstream cellular therapy assays.
Initial visual observation of the dim and diffuse objects in trypan blue stained Jurkat cells
We showed that a 2-day-old Jurkat cell sample (stored in the fridge for 2 days) stained with trypan blue displayed 3 distinct morphological populations (Figure 1) in the bright field images captured by Cellometer AutoT4.
Figure 1. Bright field cell images taken using the Cellometer Auto T4. Cells stained with trypan blue (left) and cells stained with PI (right).
- Cells that were bright, round, and plump were live cells not stained by trypan blue.
- Cells that were blue, dark, and compact are dead and stained by trypan blue.
- Large, dim, and diffuse objects were potentially dead or dying cells that were affected by trypan blue.
In addition, we have observed the increase in the diffuse objects after staining Jurkat cells, mouse splenocytes, and human PBMCs with trypan blue when cells were collected at different time points during fridge storage (Figure 2). Interestingly, Jurkat cells stained with PI did not exhibit the same morphological changes.