Cell therapy cryopreservation – delineating process parameters

At BioLife Solutions

Cell-based therapies require specialized handling to ensure transportation from the manufacturing facility to the patient is successful. To provide logistical flexibility, many current commercialization models employ a frozen cell product that can be delivered to the clinic, stored, then thawed and infused into patients on demand.

However, post-thaw viability and expansion of many cell types dramatically suffer from cryopreservation-induced stresses, collectively known as delayed-onset cell death (DOCD). As such, it is assumed that some cell types cannot be cryopreserved, and successful commercial models should be based on fresh cell culture and delivery.

Challenges of cryopreservation

The critical steps of cryopreservation overlap with manufacturing processes immediately prior to and after cell culture. As such, the efficiency and efficacy of the final therapeutic dose, as well as the manufacturing process, are directly affected by cryopreservation critical process parameters. Therefore, thorough understanding of the impact of cryopreservation on cells is vital for successful commercial manufacturing of cellular therapies.

In this study, we investigated the impact of some of the most well-known, and some of the more obscure, critical process parameters (CPPs) on post-thaw viability and proliferation in a Jurkat T cell model. Our results suggest that besides incorporating Biopreservation Best Practices for cryomedia formulation, other seemingly unimportant and irrelevant process parameters that may generally be neglected, can have a significant impact on cell viability and proliferation post-thaw.

Successful cryopreservation method detectable 24 hours post-thaw

The results suggest that, using the Standard CPPs, both CryoStor CS5 and home-brew formulation composed of PLA, 10% w/v HSA, and 5% v/v DMSO, performed similarly in preserving viability and proliferation capacity of Jurkat T cells.

However, CryoStor CS5 appeared to significantly minimize the stresses due to dilution or stochastic nucleation, as such, protecting the cells against variations in cryopreservation CPPs (or nonstandard CPPs), resulting in improved viability and count at 24 hours post-thaw and decreasing variability in the results.

The figure shows viable recovery and expansion of Jurkat T cells post-thaw assessed using a membrane integrity assay (the Annexin V Assay). (* p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001). Red asterisks (*) refer to comparison with Standard Practice results. Error bars denote SD. CS5: CryoStor® CS5; HB: Home-brew.

See full dataset in the PDF download

Bar chart showing viable recovery and expansion of Jurkat T cells post-thaw assessed using the Annexin V Assay.


Immediate post-thaw analysis of viability and count is not reflective of a successful cryopreservation process. Adverse effects of nonoptimized cryomedia may not be detectable until 24-48 hours post-thaw, attributed to cryopreservation-induced DOCD.

Incorporation of optimized cryopreservation media, on the other hand, can protect against DOCD, resulting in decreased variability and a more accurate estimation of post-thaw survival and proliferation.


Alireza Abazari, Brian J. Hawkins and Aby J. Mathew.