Cytokine analysis on a countable number of molecules from living single cells on nanofluidic devices

The Analyst ◽  
2019 ◽  
Vol 144 (24) ◽  
pp. 7200-7208 ◽  
Author(s):  
Tatsuro Nakao ◽  
Yutaka Kazoe ◽  
Emi Mori ◽  
Kyojiro Morikawa ◽  
Takemichi Fukasawa ◽  
...  
Keyword(s):  
Single Cells ◽  
Countable Number ◽  
Cytokine Analysis ◽  
Protein Molecules ◽  
Nanofluidic Devices ◽  
Cellular Processing ◽  
Nanofluidic Device ◽  
Living Single

Analysis of a countable number of protein molecules released from living single cells was realized by a micro/nanofluidic device entirely integrating cellular processing and molecular processing into pL-microchannels and fL-nanochannels, respectively.

EXTH-40. MULTIDIMENSIONAL INTERROGATION OF GLIOBLASTOMA MICROENVIRONMENT TREATMENT RESPONSE IN EXPLANT CULTURES

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi172-vi172
Author(s):  
Tala Shekarian ◽  
Ewelina Bartoszek-Kandler ◽  
Carl Zinner ◽  
Christian Schuerch ◽  
Gregor Hutter
Keyword(s):  
Checkpoint Inhibitors ◽  
Single Cells ◽  
Response Assessment ◽  
Response Analysis ◽  
Cellular Composition ◽  
Cytokine Analysis ◽  
Adaptive Immune Cells ◽  
Bioreactor Cultures ◽  
Tumor Center ◽  
Explant Cultures

Abstract The immune tumor microenvironment (iTME) of glioblastoma (GBM) contains microglial, macrophage, other myeloid cell populations and as adaptive immune cells. Recent therapeutic strategies for GBM aim at targeting iTME components to induce antitumoral immunity. A patient-tailored, ex vivo drug testing and response analysis platform would facilitate personalized therapy planning, provide insights into treatment-induced immune mechanisms in the iTME, and enable the discovery of biomarkers of response and resistance. Here, we generated patient-derived, live 3D GBM bioreactors from different tumor regions to assess iTME treatment responses to microglia modulators and immune checkpoint inhibitors. Intact GBM tissue specimens from the tumor center and periphery were cultured for 7 days in the presence or absence of anti-PD1, anti-CD47 antibodies or their combination. Tissues were analyzed by CODEX highly multiplexed microscopy using an immune-centered 54-marker panel, and changes in cytokine and chemokine levels in culture supernatants were investigated. A computational pipeline for integrative therapy response assessment was implemented. Explant cultures from n=8 IDH wt GBM were subjected to this integrative personalized analysis. Tissue integrity after 3D bioreactor cultures was comparable to tissue taken directly after surgery. FFPE CODEX workflow was feasible with adequate staining quality in bioreactor cultures. 850'000 single cells were segmented and clustered. Cellular composition between tumor center and the peripheral invasion zone differed significantly in immune phenotypes, cytokine profile and response to innate, adaptive or combinatorial local immunotherapies. Multiplexed cytokine analysis revealed IFNγ response signatures in a subset of center samples, whereas the peripheral invasion zone displayed a blunted cytokine response. This cytokine signature corresponded to cellular composition shifts within specific cellular neighborhoods. CD4 and CD8 T cells were invigorated and left their vascular niche. Our study demonstrates that local immunotherapies enable an active antitumoral immune response within the tumor center, and provides a multidimensional personalized framework for immunotherapy response assessment.

Picoliter liquid handling at gas/liquid interface by surface and geometry control in a micro-nanofluidic device

2021 ◽  
Author(s):  
Kyojiro Morikawa ◽  
Shin-ichi Murata ◽  
Y Kazoe ◽  
Kazuma Mawatari ◽  
Takehiko Kitamori
Keyword(s):  
Surface Tension ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Cell Lysis ◽  
Fused Silica ◽  
Liquid Interface ◽  
Nanofluidic Devices ◽  
Fluidic Control ◽  
Lysis Buffer ◽  
Nanofluidic Device

Abstract In micro- and nanofluidic devices, highly precise fluidic control is essential. Conventional mechanical valves in microchannels and nanochannels have size limitations, whereas hydrophobic (Laplace) valves are generally difficult to use for low-surface-tension liquids. In the present study, we developed a method for handling picoliter volumes of low-surface-tension liquids in a micro-nanofluidic device. The proposed Laplace valve is based on the pinning effect. A fused silica micro-nanofluidic device that includes a picoliter chamber whose geometry was designed to induce capillary pinning was designed and fabricated. The measured Laplace pressure of a lysis buffer (surfactant) was consistent with the calculated pressure, indicating successful fabrication and hydrophobic surface modification. The working principle of the Laplace valve was verified. The Laplace valve maintained the lysis buffer at the gas/liquid interface for 60 min, which is sufficiently long for cell lysis operations. Finally, replacement of liquids in the picoliter chamber using the valve was demonstrated. The proposed method will contribute to basic technologies for fluidic control in micro- and nanofluidic devices, and the proposed Laplace valve can be used for low-surface-tension liquids. In addition, the developed valve and picoliter chamber can be utilized for the interface in single-cell lysis, which will facilitate the development of single-cell analysis devices.

Micro/extended-nano sampling interface from a living single cell

The Analyst ◽  
2017 ◽  
Vol 142 (10) ◽  
pp. 1689-1696 ◽  
Author(s):  
L. Lin ◽  
K. Mawatari ◽  
K. Morikawa ◽  
Y. Pihosh ◽  
A. Yoshizaki ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cell Analysis ◽  
Sampling Interface ◽  
Living Single

Single-cell analysis is of increasing importance in many fields, but is challenging due to the ultra-small volumes (picoliters) of single cells.

scholarly journals Scalable integration of nano-, and microfluidics with hybrid two-photon lithography

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Oliver Vanderpoorten ◽  
Quentin Peter ◽  
Pavan K. Challa ◽  
Ulrich F. Keyser ◽  
Jeremy Baumberg ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Device Fabrication ◽  
Successful Operation ◽  
Force Microscopy ◽  
Two Photon ◽  
Nanofluidic Channels ◽  
Atomic Force ◽  
Nanofluidic Devices ◽  
Nanofluidic Device

Abstract Nanofluidic devices have great potential for applications in areas ranging from renewable energy to human health. A crucial requirement for the successful operation of nanofluidic devices is the ability to interface them in a scalable manner with the outside world. Here, we demonstrate a hybrid two photon nanolithography approach interfaced with conventional mask whole-wafer UV-photolithography to generate master wafers for the fabrication of integrated micro and nanofluidic devices. Using this approach we demonstrate the fabrication of molds from SU-8 photoresist with nanofluidic features down to 230 nm lateral width and channel heights from micron to sub-100 nm. Scanning electron microscopy and atomic force microscopy were used to characterize the printing capabilities of the system and show the integration of nanofluidic channels into an existing microfluidic chip design. The functionality of the devices was demonstrated through super-resolution microscopy, allowing the observation of features below the diffraction limit of light produced using our approach. Single molecule localization of diffusing dye molecules verified the successful imprint of nanochannels and the spatial confinement of molecules to 200 nm across the nanochannel molded from the master wafer. This approach integrates readily with current microfluidic fabrication methods and allows the combination of microfluidic devices with locally two-photon-written nano-sized functionalities, enabling rapid nanofluidic device fabrication and enhancement of existing microfluidic device architectures with nanofluidic features.

scholarly journals High-throughput single-cell TCR - pMHC dissociation rate measurements performed by an autonomous microfluidic cellular processing unit

2021 ◽  
Author(s):  
Fabien Jammes ◽  
Julien Schmidt ◽  
George Coukos ◽  
Sebastian Josef Maerkl
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Single Cell ◽  
High Throughput ◽  
Cd8 T Cells ◽  
Single Cells ◽  
Dissociation Rate ◽  
Processing Unit ◽  
Proof Of Concept ◽  
Cellular Processing

We developed an integrated microfluidic cellular processing unit (mCPU) capable of autonomously isolating single cells, perform, measure, and on-the-fly analyze cell-surface dissociation rates, followed by recovery of selected cells. We performed proof-of-concept, high- throughput single-cell experiments characterizing pMHC-TCR interactions on live CD8 T cells. The mCPU platform analyzed TCR-pMHC dissociation rates with a throughput of 50 cells per hour and hundreds of cells per run, and we demonstrate that cells can be selected, enriched, and easily recovered from the device.

scholarly journals Influence of Storage on Monodispersed Cells of Mycobacterium terrae Used for Quantitative Carrier Test prEN 14563

2003 ◽  
Vol 69 (11) ◽  
pp. 6932-6934 ◽  
Author(s):  
Eva Woelk ◽  
Peter Goroncy-Bermes ◽  
Wolfgang Sand
Keyword(s):  
Single Cells ◽  
Carrier Test ◽  
Living Single

ABSTRACT The degree of cell clumping increased with time of storage (1% cell clumps immediately after homogenization and 3 and 6.5% after 48 and 96 h of storage, respectively), and the number of living single cells decreased. Quantitative carrier tests were carried out with these cells using ortho-phthaldialdehyde (OPA) and coco fatty aminoxethylate as biocides. In contrast to OPA, with coco fatty aminoxethylate the reductions obtained with freshly homogenized mycobacteria were significantly higher (P = 0.02) than those obtained with mycobacteria kept in the refrigerator for 4 days. Therefore, it is advisable to prepare the test suspension freshly for each test.

scholarly journals Advances in Label-Free Detections for Nanofluidic Analytical Devices

Micromachines ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 885 ◽  
Author(s):  
Thu Le ◽  
Hisashi Shimizu ◽  
Kyojiro Morikawa
Keyword(s):  
Single Molecule ◽  
Detection Methods ◽  
Label Free ◽  
Practical Applications ◽  
Detection Techniques ◽  
Kinetic Information ◽  
Label Free Detection ◽  
Nanofluidic Devices ◽  
Nanofluidic Device ◽  
New Research

Nanofluidics, a discipline of science and engineering of fluids confined to structures at the 1–1000 nm scale, has experienced significant growth over the past decade. Nanofluidics have offered fascinating platforms for chemical and biological analyses by exploiting the unique characteristics of liquids and molecules confined in nanospaces; however, the difficulty to detect molecules in extremely small spaces hampers the practical applications of nanofluidic devices. Laser-induced fluorescence microscopy with single-molecule sensitivity has been so far a major detection method in nanofluidics, but issues arising from labeling and photobleaching limit its application. Recently, numerous label-free detection methods have been developed to identify and determine the number of molecules, as well as provide chemical, conformational, and kinetic information of molecules. This review focuses on label-free detection techniques designed for nanofluidics; these techniques are divided into two groups: optical and electrical/electrochemical detection methods. In this review, we discuss on the developed nanofluidic device architectures, elucidate the mechanisms by which the utilization of nanofluidics in manipulating molecules and controlling light–matter interactions enhances the capabilities of biological and chemical analyses, and highlight new research directions in the field of detections in nanofluidics.

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