In several cell therapy workflows, cryopreservation is a prevailing topic of high importance.

Does Trypan Blue Accurately Measure Viability Post-Thaw?

Cellular therapy and processing protocols stress the importance of storage conditions and minimal temperature fluctuations to achieve suitable recovery of viable and functional cells for subsequent immune assays. Freeze-thaw cycles and other manipulations stress primary cells and induce pathways of cell death. The common dye exclusion method to measure viability during cryopreservation is the trypan blue (TB) method. It is not unique to observe a 10-30% drop in viability pre- versus post-thaw based on a site’s protocol. This difference becomes more significant when samples processed and frozen at one site get thawed for revival at another.

Why does this matter?

Protocols that use trypan blue to determine viability underestimate cell death. A simple argument is size – Trypan blue is a large molecule, ~960 Da. On the other hand, Propidium iodide (PI), a red nuclear fluorescent dye, is a much smaller molecule. At ~668 Da, PI will enter compromised cell membranes more effectively than TB. The red fluorescence removes subjectivity and improves accuracy.

Tip!

Use the most sensitive method to assess viability and cell counts. Aim for high viability >80%, at least 2 million cells per aliquot -the more the better as there will always be cells loss in freeze/thaw cycles. You don’t want any surprises during revival.

Trypan Blue: A Bull in a (Freeze-Thaw) China Shop

Scientists working on primary cells also know that TB can be cytotoxic and often need to adjust their incubation time and concentration accordingly. When samples are fresh and thus highly viable, TB or the AO/PI method can be used before freezing cells in a cryopreservant solution (media + FBS+ DMSO). However, after the freeze-thaw cycle, when viability drops, TB’s cytotoxic effect can further damage cells when incubation is required, thus skewing the sample’s viability. The Nexcelom workflow using AO/PI nuclear dyes requires no incubation time and has minimal effect on cytotoxicity. Our Cellometer’s are fast which enable “real” control over % recovery calculations pre and post-thaw.

Tip!

Download our white paper to learn more about differences between AO/PI vs TB assay

Looking for Accuracy in Your Primary Cell Viability Post-Thaw?

Nexcelom instruments are uniquely designed to be accurate over a dynamic range of concentrations [10^5 cells/ml-10^7 cells/ml], and cell sizes [2 micron- 300 micron]. To test our counters pre- and post-thaw cycle, contact info@nexcelom.com to set up a free demo with one of our scientists.