Cryopreservation resulted in significant cell loss during thawing; 10,000,000 cells were cryopreserved but only 5,150,000 cells were recovered, a loss of 48.5%. Furthermore, even though HIVE collectors were loaded with equal numbers of cells per condition, fewer cryopreserved cells were recovered with the HIVE device, likely due to cell damage from cryopreservation [Figure 3A]. Other performance metrics, such as genes per cell and transcripts per cell, were similar between processing time points [Figures 3B, 3C]. Additionally, the cryopreserved cells recovered showed a higher percentage of mitochondrial reads, indicative of low-quality cells [Figure 3D].
Figure 3. A) Number of high-quality cells recovered for each condition. B) Median unique genes per cell. C) Median unique transcripts per cell. D) Percentage of mitochondrial reads per sample
Bone marrow, the spongy tissue at the center of most bones, is the primary site of hematopoiesis, the production of new blood cells. Hematopoietic stem cells give rise to progenitor cells which then differentiate into platelets (thrombocytes), red blood cells (erythrocytes), and white blood cells (leukocytes). After thresholding and filtering for high quality single-cells, a UMAP plot of single cells from all eleven HIVE devices was generated and colored by cell type [Figure 4A]. Twenty-three cell types were identified, including T cell, B cell, multiple stages of erythrocyte progenitors and fragile cell types, like neutrophils and eosinophils. Additionally, there was robust and specific expression of marker genes for each cell type [Figure 4B].
Figure 4. A) UMAP plot of high-quality single cells from eleven HIVE devices, colored by cell lineage. B) Dot plot showing the expression profile of specific marker genes (columns) for each cell type (rows)
When the cells are colored based on whether they were processed fresh (blue) or stored in a HIVE device (orange), the distribution of cells within each cell type is consistent; however, the cryopreserved samples (gray) did not retain all cell types [Figure 5A]. Approximately half of the cells recovered, from both the fresh and HIVE device stored samples, are cell types lost in the cryopreserved samples [Figure 5B]. Additionally, the relative frequency of each cell type is consistent between the fresh samples and those stored in the HIVE device for 7 days. In contrast, the cryopreserved samples have partial or complete loss of some cell types, causing the remaining cell types to be over-represented [Figure 5C]. In particular, neutrophils, eosinophils, and granulocyte/macrophage progenitors (GMPs) were depleted in the cryopreserved samples but not the samples stored in a HIVE device [Figures 5B, 5C]. The cryopreserved samples also have a cluster of poor-quality neutrophils that are not present in the d0 and d7 samples [Figure 5A].
Figure 5. A) UMAP plots from HIVE devices processed fresh (blue), after storage in the HIVE collector for 7 days (orange), or after cryopreservation for 7 days (gray). B) Pie chart showing cell classes recovered for d0, d7, and cryopreserved samples C) Relative frequencies, normalized to 1, for each cell type for HIVE devices processed fresh (blue), after storage in the HIVE collector for 7 days (orange), or after cryopreservation for 7 days (gray)