Rapid and Safe Isolation of Human Peripheral Blood B and T Lymphocytes through Spiral Microfluidic Channels

Po-Lin Chiu,Chun-Hao Chang,Ping-Hsien Tsou,Bor-Ran Li,Yu-Ling Lin

doi:10.1038/s41598-019-44677-3

Abstract

Peripheral blood lymphocytes (PBLs) are mature lymphocytes that circulate in the blood rather than being localized to organs. A reliable label-free collection approach that can viably and appropriately isolate PBLs to establish in vitro culture systems is crucial for basic research and clinical requirements. However, isolation of PBLs from whole blood is difficult, and so the development of a rapid and safe method to perform this task is required. Microfluidic technology offers opportunities that challenge the performance of macroscale methods. In this study, we proposed a simple spiral microfluidic chip for efficient and high-throughput isolation of lymphocytes from a sample with prelysed RBCs. This spiral microfluidic platform does not rely on antibodies or biological markers for labeling cells of interest while isolating lymphocytes but rather enriches B and T lymphocytes through the different physical properties that are intrinsic to lymphocytes and other blood cells. The device was used to achieve high-throughput (~1.3 × 105 cells/min) separation of lymphocytes with high viability (>95%). Compared with previous approaches, our device provided rapid, label-free, high-throughput, and safe lymphocyte separation.

Highlights

Collected blood is diluted with phosphate-buffered saline (PBS) and placed on Ficoll-Hypaque
Compared to most previous spiral inertial microfluidic devices working with syringe pumping systems[30], two pressure pumps are utilized to drive the sample and sheath buffer, which can rapidly, and steadily operate the optimized sample/sheath ratio to raise separation efficiency in a related nonmeticulous microfluidic device and enable the device to continuously dispose of a large sample. This spiral microfluidic platform takes advantage of properties of other spiral inertial www.nature.com/scientificreports microfluidic devices[31,32,33,34,35]; it does not rely on antibodies or biological markers for labeling cells of interest while isolating lymphocytes but rather enriches lymphocytes through differential physical properties that are intrinsic to lymphocytes and other blood cells
The sample was pumped into the spiral microfluidic device at a distinct pressure and collected from the five outlets

Summary

Introduction

Collected blood is diluted with phosphate-buffered saline (PBS) and placed on Ficoll-Hypaque. TM several commercial kits (e.g., RosetteSep Human Total Lymphocyte Enrichment Cocktail) have been designed to enrich lymphocytes from whole blood through negative selection[16,17,18] This method aims to remove unwanted venous blood cells by using tetrameric antibody complexes that recognize CD16, CD36, CD66b, and glycophorin A in red blood cells (RBCs). Purified lymphocytes are obtained as a highly enriched population at the interface between the plasma and buoyant density medium Another improved lymphocyte isolation method is to directly isolate lymphocytes from human whole blood through immunomagnetic selection. This approach utilizes antibody-immobilized magnetic beads to remove nonlymphocytic cells and enrich lymphocytes. The related reagent is very expensive, and immunomagnetic beads cannot be 100% removed from the final collecting cells

Methods

Results

Conclusion