Related Experiment Video
Updated: Jun 11, 2025
Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners
Published on: October 25, 2024
Cryo-PRO facilitates whole blood cryopreservation for single-cell RNA sequencing of immune cells from clinical
Alyssa K DuBois1, Pierre O Ankomah1,2, Alexis C Campbell2
1Broad Institute, Cambridge MA, USA.
Abstract:
Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) has enhanced our understanding of host immune mechanisms in small cohorts, particularly in diseases with complex and heterogeneous immune responses such as sepsis. However, PBMC isolation from blood requires technical expertise, training, and approximately two hours of onsite processing using Ficoll density gradient separation ('Ficoll') for scRNA-seq compatibility, precluding large-scale sample collection at most clinical sites. To minimize onsite processing, we developed Cryo-PRO (Cryopreservation with PBMC Recovery Offsite), a method of PBMC isolation from cryopreserved whole blood that allows immediate onsite sample cryopreservation and subsequent PBMC isolation in a central laboratory prior to sequencing. We compared scRNA-seq results from samples processed using Cryo-PRO versus standard onsite Ficoll separation in 23 patients with sepsis. Critical scRNA-seq outputs including cell substate fractions and marker genes were similar for each method across multiple cell types and substates, including an important monocyte substate enriched in patients with sepsis (Pearson correlation 0.78, p<0.001; 70% of top marker genes shared). Cryo-PRO reduced onsite sample processing time from >2 hours to <15 minutes and was reproducible across two enrollment sites, thus demonstrating potential for expanding scRNA-seq in multicenter studies of sepsis and other diseases.
Insights
Cryo-PRO enables peripheral blood mononuclear cell (PBMC) isolation from cryopreserved blood, simplifying sample processing for single-cell RNA sequencing (scRNA-seq). This method maintains data quality while significantly reducing onsite time, facilitating large-scale studies.
Area of Science:
- Immunology
- Genomics
- Clinical Research
Background:
- Single-cell RNA sequencing (scRNA-seq) provides deep insights into immune responses, particularly in complex diseases like sepsis.
- Current PBMC isolation for scRNA-seq requires extensive onsite processing (>2 hours), limiting large-scale clinical applications.
- Standard Ficoll density gradient separation is time-consuming and technically demanding at clinical sites.
Purpose of the Study:
- To develop and validate a novel method, Cryo-PRO (Cryopreservation with PBMC Recovery Offsite), for simplified PBMC isolation for scRNA-seq.
- To compare the efficacy of Cryo-PRO with standard Ficoll separation in patients with sepsis.
- To assess the feasibility of Cryo-PRO for multicenter studies by evaluating its reproducibility.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) were isolated from cryopreserved whole blood using the Cryo-PRO method.
- PBMC isolation was performed using standard Ficoll density gradient separation as a control.
- scRNA-seq data from both methods were compared for cell substate fractions and marker gene expression in 23 sepsis patients.
Main Results:
- Cryo-PRO significantly reduced onsite sample processing time to under 15 minutes.
- scRNA-seq outputs, including cell type proportions and key marker genes, were comparable between Cryo-PRO and Ficoll methods (Pearson correlation 0.78 for monocyte substate).
- The Cryo-PRO method demonstrated reproducibility across two clinical enrollment sites.
Conclusions:
- Cryo-PRO offers a robust and efficient alternative for PBMC isolation for scRNA-seq, minimizing onsite labor and time.
- This method has the potential to expand the use of scRNA-seq in large-scale, multicenter clinical studies, including sepsis research.
- Cryo-PRO facilitates broader accessibility to scRNA-seq for investigating immune heterogeneity in various diseases.
