Question: Can PBMCs be cryopreserved in whole blood without isolation for 5’ single cell sequencing?
Answer: In our limited testing, we have developed a solid cryopreservation method to preserve PBMCs in whole blood without isolating the cells first, immediately locking-in the transcriptome state. The 5’ single cell results of PBMCs isolated from the cryopreserved blood show comparable gene expression complexity, biology, and VDJ clonotypes compared to non-cryopreserved PBMCs (Day 0 fresh), as well as PBMCs preserved in the conventional practice (PBMCs isolated first and cryopreserved later).
Though differentially expressed genes (DEGs) in cryopreserved PBMCs were significant when compared to the fresh PBMCs, the PBMCs cryopreserved in whole blood show very limited DEGs when compared to the cryopreserved PBMCs.
The feasibility of cryopreserving PBMCs in whole blood offers timing options to isolate PBMCs for customers with the cryopreservation facility, who can cryopreserve and store the blood samples until they are ready to proceed. One possible application is batch process with lower costs, increased efficiency, and great flexibility.
Please note that while we have not tested this protocol for 3' Single Cell Gene Expression, the robust results of the 5' Gene Expression suggests that this protocol will likely also work with 3' experiments. We would recommend customers who are interested in using this protocol for 3' Single Cell Gene Expression perform a small pilot to ensure that the results are as expected.
Whole Blood Cryopreservation for 5’ Single Cell Sequencing
Protocol for Cryopreserving PBMCs in Whole Blood:
- 0.5M, pH 8.0 EDTA
- Cryogenic vial
- CoolCell FTS30
Steps (2.5mL of whole blood - volume can be adjusted as accordingly)
- Add 17.5 mL of CryoStor10 and 240 µL of 0.5M pH 8.0 EDTA into a 50mL conical tube.
* Blood:CryoStor10 volume ratio is 1:7
* EDTA improves biology recovery at the final concentration of 6 mM.
- Using a P1000 regular-bore pipette to add 2.5 ml blood to the tube.
- Ensure the conical tube is firmly capped and 90° invert mix 10 times.
- Use a 10mL serological pipette to draw up the mixture and dispense ~1.6mL of the blood mixture into a 2-mL cryogenic vial. 13 vials in total.
* 2-mL cryogenic vials are used at 10x, and cryogenic vials of a bigger volume like 5 mL should also work.
- Cap the sample vials firmly and place them in a controlled rate freezing container (CoolCell FTS30 & vial module).
- Transfer the controlled rate freezing container to a -80℃ freezer for overnight freezing to ensure the temperature of all vials drop to -80℃.
- Take out all sample vials from the controlled rate freezing container and place them on dry ice immediately.
* During transportation, all vials should be kept on dry ice.
- Put sample vials into a cryo freezer box and store it in a liquid nitrogen tank.
Figure 1: 5’ Barcode Rank Plots. The sample of PBMCs cryopreserved in whole blood (Cryo Blood) is comparable to the cryopreserved PBMCs and fresh PBMCs.
Figure 2: Sensitivity. At the same depth (reads per cell), the sample of PBMCs cryopreserved in whole blood (Cryo Blood) has comparable or higher sensitivity compared to the cryopreserved PBMCs and fresh PBMCs.
Figure 3: UMAP plots. The sample of PBMCs cryopreserved in whole blood (Cryo Blood) is comparable to the cryopreserved PBMCs and fresh PBMCs.
Figure 4: Selected key cell Populations. In general, the sample of PBMCs cryopreserved in whole blood has comparable cell populations to cryopreserved PBMCs and fresh PBMCs.
Figure 5: VDJ clonotypes. In the top 10 most abundant clonotypes, the PBMCs cryopreserved in whole blood are comparable to the cryopreserved PBMCs and the fresh PBMCs.
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