講演情報
[16p-K403-1]Open-type centrifugal microfluidic platform for single-cell trapping
and Granzyme B analysis
〇(P)Darcy Garza Miyazato1, Jonathan Briones1, Saori Odagiri2, Megumi Kambayashi1, Shohei Koyama3, Eiichi Tamiya2,4, Hyota Takamatsu3,5, Masato Saito1 (1.Institute for Open and Transdisciplinary Research Initiatives, Osaka University, 2.The Institute of Scientific and Industrial Research, Osaka University, 3.Graduate School of Medicine, Osaka University, 4.AIST PhotoBIO-OIL, 5.Clinical Research Center, NHO Osaka Minami Medical Center)
キーワード：
microfluidics、single-cell analysis、Granzyme B
Granzyme B (GzmB), a serine protease, serves as a fundamental biomarker for cellular immune response. Measuring the GzmB production at the single-cell level determines cellular heterogeneity within a population providing essential information beyond bulk measurements. This technique can improve patient prognosis and assess response to treatments such as immune checkpoint inhibitor (ICI) therapy. To address the need for convenient single-cell analysis in biological and clinical settings, we developed a simple and practical open-type centrifugal microfluidic device for single-cell trapping and analysis. This device allows for the convenient monitoring of Granzyme B activity at the single-cell level, and the open design allows the retrieval of captured cells for downstream applications, such as genetic analysis and protein profiling.
The microfluidic device utilizes an array of hexagonal microchambers that are partitioned by oil droplet interfaces, enabling the isolation and manipulation of individual cells. The device was fabricated using soft photolithography, and a polyimide sheet mask was used to selectively expose the microchambers to oxygen plasma enhancing the hydrophilicity and the formation of anchored droplets in the microchambers. Cells are introduced into the device through the inlet and driven into the microchambers by centrifugation force, effectively trapping single cells. Using this platform, we monitored GzmB production by measuring the fluorescence intensity of 5-FAM, a product generated upon cleavage of the FRET substrate 5-FAM/QXL®-520 (Sensolyte, Anaspec). Stimulated and non-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors were used to demonstrate the proof-of-concept (POC) of the device. Results show the difference in the GzmB activity of pre- and post-IL-2 stimulated samples, and the device revealed the variability of activity among individual cells, highlighting the heterogeneity within the population.
The microfluidic device utilizes an array of hexagonal microchambers that are partitioned by oil droplet interfaces, enabling the isolation and manipulation of individual cells. The device was fabricated using soft photolithography, and a polyimide sheet mask was used to selectively expose the microchambers to oxygen plasma enhancing the hydrophilicity and the formation of anchored droplets in the microchambers. Cells are introduced into the device through the inlet and driven into the microchambers by centrifugation force, effectively trapping single cells. Using this platform, we monitored GzmB production by measuring the fluorescence intensity of 5-FAM, a product generated upon cleavage of the FRET substrate 5-FAM/QXL®-520 (Sensolyte, Anaspec). Stimulated and non-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors were used to demonstrate the proof-of-concept (POC) of the device. Results show the difference in the GzmB activity of pre- and post-IL-2 stimulated samples, and the device revealed the variability of activity among individual cells, highlighting the heterogeneity within the population.