Impacts of cryopreservation on cell type composition and gene expression in bone marrow biopsy from multiple myeloma patients

As the development of the single cell sequencing technology, the workflow of preserving and processing cells needs to be more flexible and manageable to fit the longitudinal or complex single cell study. Consequently, considering the upstream cell cryopreservation protocol instead of using fresh cells become more common in single cell sequencing experiment. There have been several studies reported that cryopreservation protocol could largely preserve the transcriptomic profiles but it brought slight perturbations to the cells. In this case, the effect of cryopreservation on transcriptomic changes needs to be studied to understand whether there will be any bias in the downstream analysis. In this paper, we compared fresh and cryopreserved CD138+ and CD138- cells from multiple myeloma patients’ bone marrow aspirates to evaluate the impact of cryopreservation on cell population frequency and transcriptome. We found the cryopreservation protocol maintained highly consistent gene expression profiles for myeloma cells (R ≥ 0.96) and their microenvironment (R ≥ 0.9), while it slightly impacted the proportion of certain immune cell subtypes in the CD138- samples. In general, our cryopreservation protocol could well preserve bone marrow aspirate samples from multiple patients. To investigate the impact of cryopreservation on myeloma cells and their microenvironment, scRNA-seq data was used to compare the cellular composition and gene expression profiles of fresh vs. frozen cells that had been isolated from bone marrow aspirates from the six patients. Magnetic beads covalently coupled to CD138-specific antibodies were used to sort cells expressing CD138. Positive and negative CD138 cell populations were then divided into two aliquots each; one was immediately processed for scRNA-seq (fresh) and the other was cryopreserved for sequencing at a later date (frozen) **Raw data not provided due to patient privacy concerns**