Microfluidic assay for characterization of the leukocyte adhesion cascade
Inventors
Prabhakarpandian, Balabhaskar • Pant, Kapil • Sundaram, Shivshankar
Assignees
Publication Number
US-8380443-B2
Publication Date
2013-02-19
Expiration Date
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Abstract
An apparatus and method for identifying and screening for agents affecting the leukocyte adhesion cascade (LAC) encompassing rolling, adhesion and migration comprises an optically clear, plastic microfluidic chip comprising flow channels with diameters in the range of 10-500 μm. The flow channels are coated with endothelial cells and at least a portion of the flow channels contains 1-30 μm sized openings, optionally filled with a native or synthetic extracellular matrix, that allow leukocyte migration into one or more tissue spaces.
Core Innovation
An apparatus and method for identifying and screening for agents affecting the leukocyte adhesion cascade (LAC) encompassing rolling, adhesion and migration comprises an optically clear, plastic microfluidic chip comprising flow channels with diameters in the range of 10-500 μm. The lumenal walls of the flow channels are coated with endothelial cells and at least a portion of the flow channels contains 1-30 μm sized openings, optionally filled with a native or synthetic extracellular matrix, that allow leukocyte migration into one or more tissue spaces.
Current in-vitro models do not resolve rolling, adhesion and migration in a single assay: flow chambers provide physiological shear but cannot model transmigration, and Transwell and Boyden chambers do not account for fluid shear, do not provide real-time visualization of leukocyte migration, measure leukocyte migration semi-quantitatively, and are labor intensive. As a consequence, understanding of the leukocyte adhesion cascade and anti-inflammation drug development has been limited, creating a need for an assay capable of identifying and screening for agents affecting the leukocyte adhesion cascade.
In contrast with current in-vitro models, the described device resolves and facilitates direct assessment of individual steps in the leukocyte adhesion cascade including rolling, firm arrest (adhesion), spreading, and extravasation of the leukocytes into the extra-vascular tissue space. The present assay may include quantitative end point measurement, real-time visualization of cell migration, and automation of assay method steps.
Claims Coverage
The claims disclose four primary inventive features covering a method, microfluidic chip architectures, and an apparatus integrating the chip with pumping and optical detection.
Method for identifying agents that modulate leukocyte rolling, adhesion, and/or migration
Flowing a suspension of leukocytes and a solution containing an agent through an optically transparent microfluidic chip, locating and counting leukocytes in the microfluidic chip, and based upon the locations and numbers of leukocytes, determining whether the agent is modulating leukocyte rolling, adhesion, and/or migration.
Microfluidic chip architecture with non-linear, interconnected flow channels and tissue space chamber with porous walls
An optically transparent microfluidic chip comprising a plurality of non-linear, interconnected flow channels having maximum lumenal cross-sections between walls of the flow channels of between 10 and 500 microns, at least one tissue space chamber having a maximum cross-sectional dimension of between 100 μm and 1 cm, and at least a portion of the flow channels porous with pores having maximal cross-sections of between 1 and 30 microns providing fluid communication between channels and tissue space chamber.
Apparatus integrating microfluidic chip with pumping and optical detection
An apparatus comprising the microfluidic chip together with pumping means configured to move fluid from the inlet to the outlet and optical detection means configured to locate and count cells within the microfluidic chip.
Single or plural non-linear flow channel chip embodiment with porous wall and tissue space chamber
An optically transparent microfluidic chip embodiment comprising at least one non-linear flow channel having a maximum cross-section of between 10 and 500 μm and a tissue space chamber (100 μm to 1 cm) where the flow channel includes at least one porous wall with pores of 1 to 30 μm providing fluid communication between the flow channel and the tissue space chamber.
The claims cover a method for identifying modulatory agents, microfluidic chip architectures characterized by non-linear interconnected flow channels (10–500 μm) and porous channel walls (1–30 μm) communicating with tissue space chambers (100 μm–1 cm), and an apparatus that couples the chip with pumping and optical detection to locate and count leukocytes.
Stated Advantages
Resolves and facilitates direct assessment of individual steps in the leukocyte adhesion cascade including rolling, firm arrest (adhesion), spreading, and extravasation into extra-vascular tissue space.
Provides a rapid assay for anti-inflammatory markers and drugs targeted to affect the leukocyte adhesion cascade.
May include quantitative end point measurement, real-time visualization of cell migration, and automation of assay method steps.
Documented Applications
Identifying and screening for agents affecting the leukocyte adhesion cascade (LAC) encompassing rolling, adhesion and migration.
Anti-inflammatory drug development and drug screening by injection of potential cascade inhibitors to analyze effects on adhesion and migration.
Real-time visualization and quantitative measurement of leukocyte rolling, adhesion, and migration, including generating plots of cells migrated vs. local shear rate.
Measuring properties of agents such as real time circulation, stability, half-life, aggregation, and degradation within the microfluidic device.
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