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Development of a new cell isolation device FlowMagicTM
Tomoyuki Yoshida1, Yoshiaki Sakamoto1, Aya Tsuruta1
1H.U. Group Research Institute G.K., Akiruno, Tokyo, Japan.
Abstract:
Isolation of human peripheral blood mononuclear cells (PBMCs) from blood typically involves a density gradient medium during density centrifugation. The problem of increasing red blood cell (RBC) and granulocyte (GRA) contamination during PBMC isolation as the elapsed time after blood collection increases remains unresolved. As a countermeasure against RBC contamination, hemolysis treatment is available; however, these extra steps are laborious, time-consuming, and could introduce artifacts. To overcome this challenge, we developed a novel isolation device, FlowMagic™, which features a proprietary two-layer insert structure designed to prevent RBC and GRA contamination during PBMC isolation from blood. The efficacy of this method was evaluated by isolating PBMCs from donors and analyzing immune cell populations by flow cytometry. Compared to SepMate (median (Q50) = 11.0, interquartile ranges (IQR): 8.8-19.5; p < 0.01) and Lymphoprep methods (Q50 = 9.3, IQR: 6.6-13.5; p < 0.01), FlowMagic™ achieved significantly greater reduction in RBC contamination to below detectable limits (Q50 = 0.0, IQR: 0.0-0.0), with sustained efficacy observed up to 72 hours post-collection. Additionally, the FlowMagic™ method (Q50 = 2.5, IQR: 0.5-3.4, at 48 hours, median = 4.5, IQR: 2.1-10.3, at 72 hours, respectively) significantly reduced GRA contamination compared with the SepMate (Q50 = 12.0, IQR: 7.8-25.5, at 48 hours, Q50 = 27.5, IQR: 12.3-29.0, at 72 hours, respectively; p < 0.01) and Lymphoprep methods (Q50 = 10.5, IQR: 6.9-19.8, at 48 hours, Q50 = 17.5, IQR: 13.3-23.5, at 72 hours, respectively; p < 0.01) at 48 and 72 hours after blood collection. Furthermore, the recovery rates of CD3 + , CD4 + , CD8 + , CD19 + , and CD16/56 + cells in the FlowMagic™-isolated PBMCs (Q50 = 8.6, 5.9, 2.5, 1.3, and 1.9, respectively) were significantly improved compared to those in SepMate- (Q50 = 2.2, 1.5, 0.7, 0.4, and 0.5, respectively; p < 0.01) and Lymphoprep-isolated PBMCs (Q50 = 2.4, 1.5, 0.8, 0.6, and 0.8, respectively; p < 0.01), even at 48 hours after blood collection. These findings suggest that the PBMC isolation method using FlowMagic™ is advantageous in preventing RBC and GRA contamination for research, diagnostic, and clinical applications.
Insights
A new FlowMagic™ device significantly reduces red blood cell (RBC) and granulocyte (GRA) contamination during peripheral blood mononuclear cell (PBMC) isolation. This method maintains high immune cell recovery rates for up to 72 hours post-collection, improving research and diagnostic applications.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Peripheral blood mononuclear cell (PBMC) isolation is crucial for immunological research and clinical diagnostics.
- Traditional PBMC isolation methods using density gradient centrifugation are prone to contamination by red blood cells (RBCs) and granulocytes (GRAs), especially with increased time post-blood collection.
- Existing methods to mitigate contamination, like hemolysis treatment, are often laborious, time-consuming, and can introduce experimental artifacts.
Purpose of the Study:
- To develop and evaluate a novel isolation device, FlowMagic™, designed to minimize RBC and GRA contamination during PBMC isolation.
- To assess the sustained efficacy of the FlowMagic™ method over time compared to established isolation techniques.
- To determine the impact of FlowMagic™ on the recovery rates of key immune cell populations.
Main Methods:
- Development of the FlowMagic™ device featuring a proprietary two-layer insert structure for enhanced cell separation.
- Isolation of PBMCs from human blood samples using FlowMagic™, SepMate™, and Lymphoprep™ methods.
- Quantification of RBC and GRA contamination and immune cell populations (CD3+, CD4+, CD8+, CD19+, CD16/56+) via flow cytometry.
- Comparative analysis of contamination levels and cell recovery rates at various time points post-blood collection (up to 72 hours).
Main Results:
- FlowMagic™ significantly reduced RBC contamination to undetectable levels, outperforming SepMate™ and Lymphoprep™.
- The device demonstrated sustained efficacy in reducing RBC contamination up to 72 hours post-collection.
- FlowMagic™ significantly decreased GRA contamination compared to SepMate™ and Lymphoprep™ at 48 and 72 hours.
- Significantly higher recovery rates for CD3+, CD4+, CD8+, CD19+, and CD16/56+ cells were observed with FlowMagic™ compared to both SepMate™ and Lymphoprep™, even at 48 hours post-collection.
Conclusions:
- The FlowMagic™ isolation device offers a superior method for obtaining high-purity PBMCs with minimal RBC and GRA contamination.
- The method provides sustained effectiveness over extended periods post-blood collection, enhancing sample usability.
- FlowMagic™ improves immune cell recovery rates, making it a valuable tool for research, diagnostics, and clinical applications.
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