How to Improve Cryopreservation of Leukopaks and Apheresis Products

Most immunotherapies start from cells donated by either a patient or donor – most commonly in the form of apheresis products or leukopaks. Some products enter the manufacturing process directly after collection and others are cryopreserved until they are used in the manufacturing process. It has been known for years that a variety of factors influence the quality of the pre-freeze apheresis product, which will play an important role in its post-thaw outcome. For example, anticoagulant used, type of bag, temperature at which the sample is held, centrifugation speed and time are all factors that have been shown to influence the quality of the source materials. The Association for Advancement of Blood and Biotherapies (AABB) has guidelines on apheresis/leukopak quality and the factors that influence it. These guidelines can help establish workflow at the collection site. Even if an end user is not controlling all these parameters, it is important to collect this important metadata to enable retrospective analysis of preservation outcomes and the factors that adversely affect it.

Cryopreservation of Leukopaks and Apheresis Products g GraphApheresis products/leukopaks are heterogeneous cell mixtures. Many of these cell types are of clinical interest. For example, apheresis products contain populations of T-Cells (CD3+CD4+ and CD3+CD8+), B-cells, and natural killer cells. These cell types can be isolated from the heterogeneous cell population, genetically modified, and/or expanded in culture for a cell therapy product. Typically, all apheresis products/leukopaks are cryopreserved using the same cryopreservation protocol (e.g. cooling rate and composition of freezing solution). The different cell types present in a leukopak have different biological characteristics and not surprisingly, different freezing responses. A recent study (Pi et al., 2020) demonstrated that different cell subpopulations had different freezing responses when subjected to the same freezing conditions (Fig 1). CD3+CD4+ cells survived more readily than CD3+CD8+ cells, and NK cells also had a poor outcome.  

So, what do we do to improve the preservation of heterogeneous cell populations? Current methods of preservation result in depletion of the CD3+CD8+ relative to the CD3+CD4+. The Evia Bio approach is to use a computational algorithm to improve post-thaw recovery of mixed cell populations when high levels of post-thaw recovery are desired for multiple cell types. Figure 2 demonstrates an optimization strategy used to improve post-thaw recovery of both CD3+CD4+ and CD3+CD8+ subpopulations present in leukopaks. We developed an approach that reduced cell losses in the CD3+CD8+ subpopulation while still maintaining a high recovery of the CD3+CD4+ fraction of cells. This novel approach can be used to improve the preservation of other heterogeneous cell populations with different freezing responses.

Cryopreservation of Leukopaks and Apheresis Products g Graph


Pi, C.-H., Hornberger, K., Dosa, P., & Hubel, A. (2020). Understanding the freezing responses of T cells and other subsets of human peripheral blood mononuclear cells using DSMO-free cryoprotectants. Cytotherapy, 22(5), 291–300.

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