Inertial label-free sorting and chemotaxis of polymorphonuclear neutrophil in sepsis patients based on microfluidic technology_Journal of Biomedical Engineering

Authors：

GAO Chaoru ^1,2 , YANG Xiao ^1,2 , LIU Lijuan ^2,3 , WANG Yue ^2,3 , ZHU Ling ² , ZHOU Jinhua ¹ ,  LIU Yong ^1,2 ,  YANG Ke ²

1. School of Biomedical Engineering, Anhui Medical University, Hefei 230032, P. R. China;
2. Anhui Institute of Optics and Precision Mechanics, Hefei Institute of Physical Sciences, Chinese Academy of Sciences, Hefei 230031, P. R. China;
3. University of Science and Technology of China, Hefei 230026, P. R. China;

Corresponding author：

LIU Yong, Email: lywhn@sina.com; YANG Ke, Email: keyang@aiofm.ac.cn

Keywords：

Sepsis; Microfluidic chip; Polymorphonuclear neutrophil; Sorting; chemotaxis

DOI：

10.7507/1001-5515.202304002

Video：

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Reduced chemotactic migration of polymorphonuclear neutrophil (PMN) in sepsis patients leads to decreased bacterial clearance and accelerates the progression of sepsis disease. Quantification of PMN chemotaxis in sepsis patients can help characterize the immune health of sepsis patients. Microfluidic microarrays have been widely used for cell chemotaxis analysis because of the advantages of low reagent consumption, near-physiological environment, and visualization of the migration process. Currently, the study of PMN chemotaxis using microfluidic chips is mainly limited by the cumbersome cell separation operation and low throughput of microfluidic chips. In this paper, we first designed an inertial cell sorting chip to achieve label-free separation of the two major cell types by using the basic principle that leukocytes (mainly granulocytes, lymphocytes and monocytes) and erythrocytes move to different positions of the spiral microchannel when they move in the spiral microchannel under different strength of inertial force and Dean's resistance. Subsequently, in this paper, we designed a multi-channel cell migration chip and constructed a microfluidic PMN inertial label-free sorting and chemotaxis analysis platform. The inertial cell sorting chip separates leukocyte populations and then injects them into the multi-channel cell migration chip, which can complete the chemotaxis test of PMN to chemotactic peptide (fMLP) within 15 min. The remaining cells, such as monocytes with slow motility and lymphocytes that require pre-activation with proliferative culture, do not undergo significant chemotactic migration. The test results of sepsis patients (n=6) and healthy volunteers (n=3) recruited in this study showed that the chemotaxis index (CI) and migration velocity (v) of PMN from sepsis patients were significantly weaker than those from healthy volunteers. In conclusion, the microfluidic PMN inertial label-free sorting and chemotaxis analysis platform constructed in this paper can be used as a new tool for cell label-free sorting and migration studies.

Citation： GAO Chaoru, YANG Xiao, LIU Lijuan, WANG Yue, ZHU Ling, ZHOU Jinhua, LIU Yong, YANG Ke. Inertial label-free sorting and chemotaxis of polymorphonuclear neutrophil in sepsis patients based on microfluidic technology. Journal of Biomedical Engineering, 2023, 40(6): 1217-1226. doi: 10.7507/1001-5515.202304002 Copy

1.	Ellett F, Marand A L, Irimia D. Multifactorial assessment of neutrophil chemotaxis efficiency from a drop of blood. Journal of Leukocyte Biology, 2022, 111(6): 1175-1184.
2.	Yang Y, Liu L, Guo Z, et al. Investigation and assessment of neutrophil dysfunction early after severe burn injury. Burns, 2021, 47(8): 1851-1862.
3.	Sun R, Huang J, Yang Y, et al. Dysfunction of low-density neutrophils in peripheral circulation in patients with sepsis. Scientific Reports, 2022, 12(1): 685.
4.	Zhao W, Zhao H, Li M, et al. Microfluidic devices for neutrophil chemotaxis studies. Journal of Translational Medicine, 2020, 18(1): 168.
5.	Ren J, Wang N, Guo P, et al. Recent advances in microfluidics-based cell migration research. Lab on a Chip, 2022, 22(18): 3361-3376.
6.	Mcminn P H, Hind L E, Huttenlocher A, et al. Neutrophil trafficking on-a-chip: an in vitro, organotypic model for investigating neutrophil priming, extravasation, and migration with spatiotemporal control. Lab on a Chip, 2019, 19(21): 3697-3705.
7.	Hu C, Liu J, Chen H, et al. Microfluidic platforms for gradient generation and its applications. Biochemistry & Analytical Biochemistry, 2017, 6(2): 100320.
8.	李慧来, 杨逍, 吴晓松, 等. 多通道微流控芯片的设计、仿真及细胞迁移应用研究. 生物医学工程学杂志, 2022, 39(1): 128-138.
9.	Ellett F, Jorgensen J, Marand A L, et al. Diagnosis of sepsis from a drop of blood by measurement of spontaneous neutrophil motility in a microfluidic assay. Nat Biomed Eng, 2018, 2(4): 207-214.
10.	Ren X, Getschman A E, Hwang S, et al. Investigations on T cell transmigration in a human skin-on-chip (SoC) model. Lab on a Chip, 2021, 21(8): 1527-1539.
11.	Yang K, Wu J, Peretz-soroka H, et al. Mkit: a cell migration assay based on microfluidic device and smartphone. Biosensors and Bioelectronics, 2018, 99: 259-267.
12.	Berthier E, Surfus J, Verbsky J, et al. An arrayed high-content chemotaxis assay for patient diagnosis. Integrative Biology, 2010, 2(11-12): 630-638.
13.	Wu J, Kumar-kanojia A, Hombach-klonisch S, et al. A radial microfluidic platform for higher throughput chemotaxis studies with individual gradient control. Lab on a Chip, 2018, 18(24): 3855-3864.
14.	Yang K, Peretz-Soroka H, Wu J, et al. Fibroblast growth factor 23 weakens chemotaxis of human blood neutrophils in microfluidic devices. Scientific Reports, 2017, 7(1): 3100.
15.	Jimenez-Valdes R J, Rodriguez-Moncayo R, Cedillo-Alcantar D F, et al. Massive parallel analysis of single cells in an integrated microfluidic platform. Analytical Chemistry, 2017, 89(10): 5210-5220.
16.	Bhattacharjee N, Folch A. Large-scale microfluidic gradient arrays reveal axon guidance behaviors in hippocampal neurons. Microsystems & Nanoengineering, 2017, 3: 17003.
17.	Agrawal N, Toner M, Irimia D. Neutrophil migration assay from a drop of blood. Lab on a Chip, 2008, 8(12): 2054-2061.
18.	Butler K L, Ambravaneswaran V, Agrawal N, et al. Burn injury reduces neutrophil directional migration speed in microfluidic devices. PloS one, 2010, 5(7): e11921.
19.	Hoang A N, Jones C N, Dimisko L, et al. Measuring neutrophil speed and directionality during chemotaxis, directly from a droplet of whole blood. Technology, 2013, 1(1): 49.
20.	Sackmann E K H, Berthier E, Schwantes E A, et al. Characterizing asthma from a drop of blood using neutrophil chemotaxis. Proc Natl Acad Sci U S A, 2014, 111(16): 5813-5818.
21.	Sackmann E K, Berthier E, Young E W, et al. Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays. Blood, 2012, 120(14): e45-e53.
22.	Jundi B, Ryu H, Lee D H, et al. Leukocyte function assessed via serial microlitre sampling of peripheral blood from sepsis patients correlates with disease severity. Nature Biomedical Engineering, 2019, 3(12): 961-973.
23.	Jeon H, Lee D H, Jundi B, et al. Fully automated, sample-to-answer leukocyte functional assessment platform for continuous sepsis monitoring via microliters of blood. ACS Sensors, 2021, 6(7): 2747-2756.
24.	Zeming K K, Lu R, Woo K L, et al. Multiplexed single-cell leukocyte enzymatic secretion profiling from whole blood reveals patient-specific immune signature. Analytical Chemistry, 2021, 93(10): 4374-4382.
25.	Hou H W, Petchakup C, Tay H M, et al. Rapid and label-free microfluidic neutrophil purification and phenotyping in diabetes mellitus. Scientific Reports, 2016, 6: 29410.
26.	Jiang F, Xiang N. Integrated microfluidic handheld cell sorter for high-throughput label-free malignant tumor cell sorting. Analytical Chemistry, 2022, 94(3): 1859-1866.
27.	Vona G, Sabile A, Louha M, et al. Isolation by size of epithelial tumor cells: a new method for the immunomorphological and molecular characterization of circulating tumor cells. The American Journal of Pathology, 2000, 156(1): 57-63.
28.	Zabaglo L, Ormerod M G, Parton M, et al. Cell filtration-laser scanning cytometry for the characterisation of circulating breast cancer cells. Cytometry A, 2003, 55(2): 102-108.
29.	Ward P A. Chemotaxis of mononuclear cells. The Journal of Experimental Medicine, 1968, 128(5): 1201-1221.
30.	Wilkinson P C, Komai-Koma M, Newman I. Locomotion and chemotaxis of lymphocytes. Autoimmunity, 1997, 26(1): 55-72.
31.	Seo J, Lean M H, Kole A. Membrane-free microfiltration by asymmetric inertial migration. Applied Physics Letters, 2007, 91: 033901.
32.	Bhagat A A, Hou H W, Li L D, et al. Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation. Lab on a Chip, 2011, 11(11): 1870-1878.
33.	Di Carlo D, Irimia D, Tompkins R G, et al. Continuous inertial focusing, ordering, and separation of particles in microchannels. Proc Natl Acad Sci U S A, 2007, 104(48): 18892-18897.
34.	Di Carlo D. Inertial microfluidics. Lab on a Chip, 2009, 9(21): 3038-3046.
35.	Bhagat A A S, Kuntaegowdanahalli S S, Papautsky I. Continuous particle separation in spiral microchannels using dean flows and differential migration. Lab on a Chip, 2008, 8(11): 1906-1914.
36.	Ookawara S, Street D, Ogawa K. Numerical study on development of particle concentration profiles in a curved microchannel. Chemical Engineering Science, 2006, 61(11): 3714-3724.
37.	Abdulla A, Liu W, Gholamipour-Shirazi A, et al. High-throughput isolation of circulating tumor cells using cascaded inertial focusing microfluidic channel. Analytical Chemistry, 2018, 90(7): 4397-4405.
38.	Yang X, Gao C, Liu Y, et al. Simplified cell magnetic isolation assisted SC2 chip to realize “sample in and chemotaxis out”: validated by healthy and T2DM patients’ neutrophils. Micromachines, 2022, 13(11): 1820.
39.	Wu J, Hillier C, Komenda P, et al. An all-on-chip method for testing neutrophil chemotaxis induced by fMLP and COPD patient’s sputum. Technology, 2016, 4(2): 104-109.
40.	Alves-Filho J C, Freitas A, Souto F O, et al. Regulation of chemokine receptor by Toll-like receptor 2 is critical to neutrophil migration and resistance to polymicrobial sepsis. Proc Natl Acad Sci U S A, 2009, 106(10): 4018-4023.
41.	Blackwood R A, Hartiala K T, Kwoh E E, et al. Unidirectional heterologous receptor desensitization between both the fMLP and C5a receptor and the IL-8 receptor. Journal of Leukocyte Biology, 1996, 60(1): 88-93.
42.	Coles R B, Ranney R R, Freer R J, et al. Thermal regulation of FMLP receptors on human neutrophils. Journal of Leukocyte Biology, 1989, 45(6): 529-537.
43.	Yang K, Wu J, Xu G, et al. A dual-docking microfluidic cell migration assay (D2-Chip) for testing neutrophil chemotaxis and the memory effect. Integrative Biology, 2017, 9(4): 303-312.

1. Ellett F, Marand A L, Irimia D. Multifactorial assessment of neutrophil chemotaxis efficiency from a drop of blood. Journal of Leukocyte Biology, 2022, 111(6): 1175-1184.
2. Yang Y, Liu L, Guo Z, et al. Investigation and assessment of neutrophil dysfunction early after severe burn injury. Burns, 2021, 47(8): 1851-1862.
3. Sun R, Huang J, Yang Y, et al. Dysfunction of low-density neutrophils in peripheral circulation in patients with sepsis. Scientific Reports, 2022, 12(1): 685.
4. Zhao W, Zhao H, Li M, et al. Microfluidic devices for neutrophil chemotaxis studies. Journal of Translational Medicine, 2020, 18(1): 168.
5. Ren J, Wang N, Guo P, et al. Recent advances in microfluidics-based cell migration research. Lab on a Chip, 2022, 22(18): 3361-3376.
6. Mcminn P H, Hind L E, Huttenlocher A, et al. Neutrophil trafficking on-a-chip: an in vitro, organotypic model for investigating neutrophil priming, extravasation, and migration with spatiotemporal control. Lab on a Chip, 2019, 19(21): 3697-3705.
7. Hu C, Liu J, Chen H, et al. Microfluidic platforms for gradient generation and its applications. Biochemistry & Analytical Biochemistry, 2017, 6(2): 100320.
8. 李慧来, 杨逍, 吴晓松, 等. 多通道微流控芯片的设计、仿真及细胞迁移应用研究. 生物医学工程学杂志, 2022, 39(1): 128-138.
9. Ellett F, Jorgensen J, Marand A L, et al. Diagnosis of sepsis from a drop of blood by measurement of spontaneous neutrophil motility in a microfluidic assay. Nat Biomed Eng, 2018, 2(4): 207-214.
10. Ren X, Getschman A E, Hwang S, et al. Investigations on T cell transmigration in a human skin-on-chip (SoC) model. Lab on a Chip, 2021, 21(8): 1527-1539.
11. Yang K, Wu J, Peretz-soroka H, et al. Mkit: a cell migration assay based on microfluidic device and smartphone. Biosensors and Bioelectronics, 2018, 99: 259-267.
12. Berthier E, Surfus J, Verbsky J, et al. An arrayed high-content chemotaxis assay for patient diagnosis. Integrative Biology, 2010, 2(11-12): 630-638.
13. Wu J, Kumar-kanojia A, Hombach-klonisch S, et al. A radial microfluidic platform for higher throughput chemotaxis studies with individual gradient control. Lab on a Chip, 2018, 18(24): 3855-3864.
14. Yang K, Peretz-Soroka H, Wu J, et al. Fibroblast growth factor 23 weakens chemotaxis of human blood neutrophils in microfluidic devices. Scientific Reports, 2017, 7(1): 3100.
15. Jimenez-Valdes R J, Rodriguez-Moncayo R, Cedillo-Alcantar D F, et al. Massive parallel analysis of single cells in an integrated microfluidic platform. Analytical Chemistry, 2017, 89(10): 5210-5220.
16. Bhattacharjee N, Folch A. Large-scale microfluidic gradient arrays reveal axon guidance behaviors in hippocampal neurons. Microsystems & Nanoengineering, 2017, 3: 17003.
17. Agrawal N, Toner M, Irimia D. Neutrophil migration assay from a drop of blood. Lab on a Chip, 2008, 8(12): 2054-2061.
18. Butler K L, Ambravaneswaran V, Agrawal N, et al. Burn injury reduces neutrophil directional migration speed in microfluidic devices. PloS one, 2010, 5(7): e11921.
19. Hoang A N, Jones C N, Dimisko L, et al. Measuring neutrophil speed and directionality during chemotaxis, directly from a droplet of whole blood. Technology, 2013, 1(1): 49.
20. Sackmann E K H, Berthier E, Schwantes E A, et al. Characterizing asthma from a drop of blood using neutrophil chemotaxis. Proc Natl Acad Sci U S A, 2014, 111(16): 5813-5818.
21. Sackmann E K, Berthier E, Young E W, et al. Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays. Blood, 2012, 120(14): e45-e53.
22. Jundi B, Ryu H, Lee D H, et al. Leukocyte function assessed via serial microlitre sampling of peripheral blood from sepsis patients correlates with disease severity. Nature Biomedical Engineering, 2019, 3(12): 961-973.
23. Jeon H, Lee D H, Jundi B, et al. Fully automated, sample-to-answer leukocyte functional assessment platform for continuous sepsis monitoring via microliters of blood. ACS Sensors, 2021, 6(7): 2747-2756.
24. Zeming K K, Lu R, Woo K L, et al. Multiplexed single-cell leukocyte enzymatic secretion profiling from whole blood reveals patient-specific immune signature. Analytical Chemistry, 2021, 93(10): 4374-4382.
25. Hou H W, Petchakup C, Tay H M, et al. Rapid and label-free microfluidic neutrophil purification and phenotyping in diabetes mellitus. Scientific Reports, 2016, 6: 29410.
26. Jiang F, Xiang N. Integrated microfluidic handheld cell sorter for high-throughput label-free malignant tumor cell sorting. Analytical Chemistry, 2022, 94(3): 1859-1866.
27. Vona G, Sabile A, Louha M, et al. Isolation by size of epithelial tumor cells: a new method for the immunomorphological and molecular characterization of circulating tumor cells. The American Journal of Pathology, 2000, 156(1): 57-63.
28. Zabaglo L, Ormerod M G, Parton M, et al. Cell filtration-laser scanning cytometry for the characterisation of circulating breast cancer cells. Cytometry A, 2003, 55(2): 102-108.
29. Ward P A. Chemotaxis of mononuclear cells. The Journal of Experimental Medicine, 1968, 128(5): 1201-1221.
30. Wilkinson P C, Komai-Koma M, Newman I. Locomotion and chemotaxis of lymphocytes. Autoimmunity, 1997, 26(1): 55-72.
31. Seo J, Lean M H, Kole A. Membrane-free microfiltration by asymmetric inertial migration. Applied Physics Letters, 2007, 91: 033901.
32. Bhagat A A, Hou H W, Li L D, et al. Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation. Lab on a Chip, 2011, 11(11): 1870-1878.
33. Di Carlo D, Irimia D, Tompkins R G, et al. Continuous inertial focusing, ordering, and separation of particles in microchannels. Proc Natl Acad Sci U S A, 2007, 104(48): 18892-18897.
34. Di Carlo D. Inertial microfluidics. Lab on a Chip, 2009, 9(21): 3038-3046.
35. Bhagat A A S, Kuntaegowdanahalli S S, Papautsky I. Continuous particle separation in spiral microchannels using dean flows and differential migration. Lab on a Chip, 2008, 8(11): 1906-1914.
36. Ookawara S, Street D, Ogawa K. Numerical study on development of particle concentration profiles in a curved microchannel. Chemical Engineering Science, 2006, 61(11): 3714-3724.
37. Abdulla A, Liu W, Gholamipour-Shirazi A, et al. High-throughput isolation of circulating tumor cells using cascaded inertial focusing microfluidic channel. Analytical Chemistry, 2018, 90(7): 4397-4405.
38. Yang X, Gao C, Liu Y, et al. Simplified cell magnetic isolation assisted SC2 chip to realize “sample in and chemotaxis out”: validated by healthy and T2DM patients’ neutrophils. Micromachines, 2022, 13(11): 1820.
39. Wu J, Hillier C, Komenda P, et al. An all-on-chip method for testing neutrophil chemotaxis induced by fMLP and COPD patient’s sputum. Technology, 2016, 4(2): 104-109.
40. Alves-Filho J C, Freitas A, Souto F O, et al. Regulation of chemokine receptor by Toll-like receptor 2 is critical to neutrophil migration and resistance to polymicrobial sepsis. Proc Natl Acad Sci U S A, 2009, 106(10): 4018-4023.
41. Blackwood R A, Hartiala K T, Kwoh E E, et al. Unidirectional heterologous receptor desensitization between both the fMLP and C5a receptor and the IL-8 receptor. Journal of Leukocyte Biology, 1996, 60(1): 88-93.
42. Coles R B, Ranney R R, Freer R J, et al. Thermal regulation of FMLP receptors on human neutrophils. Journal of Leukocyte Biology, 1989, 45(6): 529-537.
43. Yang K, Wu J, Xu G, et al. A dual-docking microfluidic cell migration assay (D2-Chip) for testing neutrophil chemotaxis and the memory effect. Integrative Biology, 2017, 9(4): 303-312.

Journal of Biomedical Engineering

Inertial label-free sorting and chemotaxis of polymorphonuclear neutrophil in sepsis patients based on microfluidic technology

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