Complex chemoattractive and chemorepellent Kit signals revealed by direct imaging of murine mast cells in microfluidic gradient chambers

Abstract

Besides its cooperating effects on stem cell proliferation and survival, Kit ligand (KL) is a potent chemotactic protein. While transwell assays permit studies of the frequency of migrating cells, the lack of direct visualization precludes dynamic chemotaxis studies. In response, we utilize microfluidic chambers that enable direct observation of murine bone marrow-derived mast cells (BMMC) within stable KL gradients. Using this system, individual Kit+ BMMC were quantitatively analyzed for migration speed and directionality during KL-induced chemotaxis. Our results indicated a minimum activating threshold of ~3 ng ml−1 for chemoattraction. Analysis of cells at KL concentrations below 3 ng ml−1 revealed a paradoxical chemorepulsion, which has not been described previously. Unlike chemoattraction, which occurred continuously after an initial time lag, chemorepulsion occurred only during the first 90 minutes of observation. Both chemoattraction and chemorepulsion required the action of G-protein coupled receptors (GPCR), as treatment with pertussis toxin abrogated directed migration. These results differ from previous studies of GPCR-mediated chemotaxis, where chemorepulsion occurred at high ligand concentrations. These data indicate that Kit-mediated chemotaxis is more complex than previously understood, with the involvement of GPCRs in addition to the Kit receptor tyrosine kinase and the presence of both chemoattractive and chemorepellent phases.

Microfluidic analysis of extracellular matrix-bFGF crosstalk on primary human myoblast chemoproliferation, chemokinesis, and chemotaxis

“…The CCC is flanked by two arrays of micro-capillaries, which increase fluidic resistance and prevent fluid flow and the generation of shear forces within the CCC 28 . The absence of fluid flow within the CCC makes it easy to adapt the device to observe the behavior of loosely adherent cells 29,35 or cells in 3D matrices 36,37 . Channels on either side of the arrays are perfused with medium, such that the direction of fluid flow runs perpendicular to the micro-capillaries.…”

“…Another common challenge in the interpretation of data from Transwell experiments is that chemokinesis and chemoproliferation may be incorrectly interpreted as chemotaxis 27 . Thus, to independently evaluate bFGF-induced chemo kinesis (an increase in random motility ) and chemo taxis (an increase in directional migration ) in myoblasts, we utilized a microfluidic assay capable of producing a stable, linear concentration gradient 28,29 . In contrast to Transwell assays, which preclude direct visualization of cell migration, this microfluidic assay allows quantitative, single-cell measurements of migration speed and direction through the use of time-lapse microscopy.…”

Small-molecule axon-polarization studies enabled by a shear-free microfluidic gradient generator

“…The diffusion coefficient of fluorescein (MW 372), in water, D fluorescein/water = 6.4 × 10 −6 cm 2 s −1 66 , and in crosslinked PEG-DA, D fluorescein/PEG = 1.5 × 10 −6 cm 2 s −1 , was used in all simulations. D fluorescein/PEG was estimated based on the reported diffusion coefficient of casamino acid (MW 250, 1.5 ± 0.5 × 10 −6 cm 2 s −1 27 ) and 2-NBDG (MW 342, 1.5 × 10 −6 cm 2 s −1 40 ) in 20% PEG-DA (MW 575).…”

“…An alternative approach modified the original serpentine device designed by Whitesides et al 19,21 by layering it on top of a series of microwells, which creates a controllable gradient without the high shear stress. Other strategies use increased fluidic resistance to reduce shear, by designing a gradient-generating region with a significantly smaller height than the main channels 30–34 or by separating the gradient-generating region from the main channels using microcapillaries 35–40 . Membranes 10,41–43 and hydrogels 44–52 have also been incorporated into gradient-generating devices to increase fluidic resistance.…”

Integrative Utilization of Microenvironments, Biomaterials and Computational Techniques for Advanced Tissue Engineering

“…Bone marrow derived mast cells (BMMCs) in the highest Kit ligand absolute concentrations (quadrant 1), showed cytoskeletal protrusions that can be commonly observed in Fig. 3C [129]. These protrusions often form long filopodia, whose action is known to be critical in cellular migration.…”