(M1030-06-36) Stabilization of Human Cells for RNA Extraction Using Capillary-Mediated Vitrification Process

Purpose: The cells of human, animal or bacteria, viruses, bodily fluids, or DNA/RNA, are collected, stored, and distributed throughout the globe for diagnostics, research, and other biobanking related activities. These biological samples typically need to be refrigerated or frozen to maintain the stability, and function of relevant biomarkers, including RNA and DNA. The requirement of cold storage adds significant logistical complexity and cost to these workflows. Development of new preservation technologies that aid ambient storage and delivery of these materials could eradicate the safety concerns associated with shipping materials on dry ice, minimize shipping costs by reducing package size and weight, eliminate the need for shipment by air, and reduce the risk of material loss both in storage and delivery due to temperature excursions. We innovated a novel preservation method called capillary‑mediated vitrification (CMV) and have shown that it can efficiently preserve long-term mRNA integrity and function at both ambient and elevated storage conditions. Here we hypothesized that the CMV process can be adaptable to preserve mammalian cells RNA, which can be used for sequencing and biobanking applications.

Methods: The CMV process was executed by mixing 5x10⁶ Jurkat cells with an excipient buffer and adding the mixture to a porous scaffold. The loaded scaffolds were placed into our vitrification chamber and dried for 30 min. After drying, the CMV-stabilized Jurkat cells were packed in Mylar pouches were stored at 55°C for 3 days. Cells resuspended in buffer were stored at 55°C for 3 days as a negative control. Freshly cultured Jurkat cells were used as positive control. Following storage, total RNA was isolated, and the nucleic acid concentration was analyzed by Nanodrop. The RNA extracted from positive control and CMV-stabilized Jurkat cells was utilized to screen 18-tumor suppressor genes transcripts per million, percent spliced in index, and exon coverage profile using RNA-sequence analyses.

Results: To assess whether the CMV process could be used as an alternative to frozen storage to maintain cellular RNA recovery and integrity, CMV-stabilized Jurkat cells were stored under accelerated stress conditions at 55 °C for 3 days. After storage at 55°C for 3 days, the CMV-stabilized Jurkat cells had slightly lower total RNA recovery and RNA-integrity number, but exhibited a similar band integrity to that of the positive control cells. In contrast, the cells stored at 55°C in the buffer had little RNA recovery with no visible band observed. Importantly, the CMV-stabilized Jurkat cells showed similar gene transcripts, percent spliced in index, and exon coverage profile for 18 tumor suppressor genes in an RNA-sequence analysis when compared to the freshly cultured cells.

Conclusion: The study results indicate that the CMV process improved the stability of Jurkat cellular-RNA, and enabled robust gene expression profiling even after storage under extreme conditions. The study suggests that the CMV technology has the potential to enable ambient storage and shipping of human cells for molecular diagnostic and biobanking related applications.

Acknowledgements: Our company holds the license for the Capillary Mediated vitrification patent with the purpose of commercializing the technology. Pravansu Mohanty has a financial interest in this technology.

Quantity and quality of total RNA isolated from CMV-stabilized and stressed, stressed cells stored in PBS, and positive control Jurkat cells. (A) RNA yield per 5 million cells; (B) Calculated RIN values; and (C) Representative RIN electrophoretic analysis of RNA integrity. Data represent the mean value of six to eight samples ± SEM. (CMV: Capillary-mediated vitrification, RIN: RNA integrity number, SEM: Standard error mean)

Expression heatmap of 18-tumor suppressor genes transcripts per million reads for CMV-stabilized and stressed, and freshly cultured Jurkat cells (n= 6 to 8). (CMV: Capillary-mediated vitrification)

Percent spliced in index heatmap of 18-tumor suppressor genes for CMV-stabilized and stressed, and freshly cultured Jurkat cells (n= 6 to 8). (CMV: Capillary-mediated vitrification)