Stability testing of HPCs and MNCs from apheresis products

Abstract

Background

Hematopoietic progenitor cells (HPCs) and mononuclear cells (MNCs) are critical components of cell-based therapies, including bone marrow transplantation and regenerative treatments. Evaluation of the characteristics of these products during collection, storage, and transport is essential for maintaining cell viability and functionality. In this study, we evaluated the functional and molecular stability of samples collected for the evaluation of fresh HPC and MNC products. The samples stored at 4 °C for up to 4 days and were evaluated using white blood cell (WBC) counts, flow cytometry, and bulk RNA sequencing (RNA-seq) across five time points.

Methods

HPC samples from seven products (June–December 2022) and MNC samples from six products (October 2022–August 2023) were analyzed on days 0 through 4 after collection. WBC counts were measured, and viability was assessed using 7-AAD staining and flow cytometry. HPC samples were stained with antibodies against CD34, CD3, CD19, CD56, CD14, CD16, CD15, and CD45, while MNC samples were stained with antibodies directed to CD3, CD4, CD8, CD19, CD56, CD14, CD16, CD15, and CD45. Total RNA was isolated from each sample and subjected to bulk RNA-seq to assess transcriptomic changes during storage.

Results

While WBC counts varied between products, no significant differences were observed across time points within individual products. Flow cytometry markers remained relatively stable over time in both HPC and MNC samples, although greater variability was observed in HPCs. A modest decrease in lymphocyte percentages was noted at later time points, primarily driven by a reduction in CD3+ cells; however, these changes were not statistically significant. Cell viability declined significantly over time within individual products and showed inter-product variability. RNA-seq analysis revealed stable gene expression profiles in MNC samples across all time points. In contrast, HPC samples exhibited notable transcriptomic changes as early as day 1 of storage at 4 °C, indicating greater molecular instability.

Conclusion

WBC counts and flow cytometry markers remain stable for up to 3 days in samples collected from fresh HPC and MNC products when stored at 4 °C, although cell viability progressively declines. However, RNA-seq data reveal early transcriptomic changes in HPC samples, suggesting that immediate evaluation of these samples is critical to preserve their molecular integrity and functionality. These findings support the feasibility of delayed phenotypic analysis but emphasize the need for prompt molecular assays in HPC-based applications.