Wanxin Wang1, Lolita Penland1,2, Ozgun Gokce3,4, Derek Croote1, Stephen R Quake5,6,7. 1. Department of Bioengineering, Stanford University, James H Clark Center, E300, 318 Campus Drive, Stanford, CA, 94305, USA. 2. Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA. 3. Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, 94305, USA. 4. Institute for Stroke and Dementia Research, Klinikum der Universität München, Ludwig Maximilians Universität LMU, 81377, Munich, Germany. 5. Department of Bioengineering, Stanford University, James H Clark Center, E300, 318 Campus Drive, Stanford, CA, 94305, USA. quake@stanford.edu. 6. Department of Applied Physics, Stanford University, James H Clark Center, E300, 318 Campus Drive, Stanford, CA, 94305, USA. quake@stanford.edu. 7. Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA. quake@stanford.edu.
Abstract
BACKGROUND: High-fidelity preservation strategies for primary tissues are in great demand in the single cell RNAseq community. A reliable method would greatly expand the scope of feasible multi-site collaborations and maximize the utilization of technical expertise. When choosing a method, standardizability and fidelity are important factors to consider due to the susceptibility of single-cell RNAseq analysis to technical noise. Existing approaches such as cryopreservation and chemical fixation are less than ideal for failing to satisfy either or both of these standards. RESULTS: Here we propose a new strategy that leverages preservation schemes developed for organ transplantation. We evaluated the strategy by storing intact mouse kidneys in organ transplant preservative solution at hypothermic temperature for up to 4 days (6 h, 1, 2, 3, and 4 days), and comparing the quality of preserved and fresh samples using FACS and single cell RNAseq. We demonstrate that the strategy effectively maintained cell viability, transcriptome integrity, cell population heterogeneity, and transcriptome landscape stability for samples after up to 3 days of preservation. The strategy also facilitated the definition of the diverse spectrum of kidney resident immune cells, to our knowledge the first time at single cell resolution. CONCLUSIONS: Hypothermic storage of intact primary tissues in organ transplant preservative maintains the quality and stability of the transcriptome of cells for single cell RNAseq analysis. The strategy is readily generalizable to primary specimens from other tissue types for single cell RNAseq analysis.
BACKGROUND: High-fidelity preservation strategies for primary tissues are in great demand in the single cell RNAseq community. A reliable method would greatly expand the scope of feasible multi-site collaborations and maximize the utilization of technical expertise. When choosing a method, standardizability and fidelity are important factors to consider due to the susceptibility of single-cell RNAseq analysis to technical noise. Existing approaches such as cryopreservation and chemical fixation are less than ideal for failing to satisfy either or both of these standards. RESULTS: Here we propose a new strategy that leverages preservation schemes developed for organ transplantation. We evaluated the strategy by storing intact mouse kidneys in organ transplant preservative solution at hypothermic temperature for up to 4 days (6 h, 1, 2, 3, and 4 days), and comparing the quality of preserved and fresh samples using FACS and single cell RNAseq. We demonstrate that the strategy effectively maintained cell viability, transcriptome integrity, cell population heterogeneity, and transcriptome landscape stability for samples after up to 3 days of preservation. The strategy also facilitated the definition of the diverse spectrum of kidney resident immune cells, to our knowledge the first time at single cell resolution. CONCLUSIONS: Hypothermic storage of intact primary tissues in organ transplant preservative maintains the quality and stability of the transcriptome of cells for single cell RNAseq analysis. The strategy is readily generalizable to primary specimens from other tissue types for single cell RNAseq analysis.
Entities:
Keywords:
Hypothermic preservation; Kidney resident immune cells; Organ transplant preservation; Organ transplant preservative; Primary tissue preservation without dissociation; Single cell RNAseq; Transcriptome variability analysis
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