Impact of pre- and post-thaw processing on recovery and fitness of cord blood mononuclear cells as starting material for cell therapy.

Abstract

Background: Cord blood units (CBUs) are a well-established source for hematopoietic stem cell transplantation, but key challenges such as post-thaw recovery and functional integrity remain to be addressed for their broader use in emerging cell therapy applications.This study evaluates the impact of pre-cryopreservation mononuclear cell isolation and compares four post-thaw processing methods - Wash-only, Density Gradient, Beads (CD15/CD235 depletion), and EasySep™ Direct Human PBMC Isolation Kit - on CBMC recovery, purity, and functional fitness.

Study design and methods: CBUs were processed either by standard volume reduction or by mononuclear cell isolation prior to cryopreservation. Colony-forming Units, metabolic activity, and Live, Apoptosis-Negative (LAN) assays were performed pre-freeze and post-thaw to compare cell fitness across these two pre-cryopreservation approaches. Separately, post-thaw CBMCs were isolated from volume-reduced CBUs using the four different methods, and outcomes were assessed by flow cytometry for immune subset recovery on day 0, as well as T cell proliferation and cell fitness with the apoptosis assay after five days of culture.

Results: Pre-cryopreservation mononuclear cell isolation did not improve post-thaw CBMC recovery or function compared to standard volume-reduced units. Beads and PBMC Isolation Kit achieved the highest depletion, while Wash-Only retained the lowest levels of purity but the highest CBMC yield. PBMC Isolation Kit - processed samples showed the highest percentage of viable LAN cells on Day 0, whereas the Beads method best preserved viability over five days of stimulation. The LAN assay, previously validated only for fresh cells, was successfully applied to post-thaw CBUs. Notably, PBMC Isolation Kit significantly depleted CD14+ cells, which correlated with reduced T cell proliferation.

Discussion: These findings highlight trade-offs between purity, recovery, and functional outcomes in CBU processing. The choice of method should be application specific, particularly when antigen presentation or long-term viability is required. This study provides a framework for optimizing post-thaw processing to enhance CBU suitability for cell therapy.