chemotaxis, transmigration and invasion assays

Overview

Cell attachment to the microcapillary wall as a result of sensing of chemotactic molecules, subsequent transmigration through the endothelial cell layer and invasion into the underlying tissue are all key steps in cell-cell signaling and involved in inflammation and therapeutic areas such as oncology and cardiovascular diseases (e.g. atherosclerosis).. These experimental conditions can easily be studied using Cellix's microfluidic pumps and biochips.  The dimensions of the biochips can mimic those of human capillaries thereby enabling researchers to simulate in vivo microenvironments. A key additional benefit of using microfluidics is the reduction in sample volume required for experiments.  This is particularly important when using whole blood.  Cellix's 8-channel Mirus Evo Nanopump enables researchers to conduct up to 8 assays simultaneously in microfluidic biochips while single assays may be conducted using the ExiGo pump.  The microcapillary walls of Cellix's biochips may be pre-coated with fibronectin to promote culturing of endothelial cells (e.g. HUVECs, HMVECs, HLMECs) via the Kima pump, resulting in a set-up for studying chemotaxis, transmigration and invasion assays.

Assays:  

  • cell chemotaxis, transmigration and invasion assays.
  • cell observation with brightfield, phase contrast and immunostaining microscopy (all biochips)
  • T-cell-endothelial; neutrophil-endothelial; PBMC-endothelial; monocyte-endothelial; eosinophil-endothelial cell interaction studies
  • investigation of anti-inflammatory, oncology and cardiovascular therapies

 

Assay Examples:

 

Proteinase 3 Contributes to Transendothelial Migration of NB1-Positive Neutrophils

  • NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. 
  • Aim:  To investigate NB1-PR3 interactions and their role in aiding neutorphil diapedesis.
  • Neutrophils were isolated from healthy donors and for some experiments pretreated with inhibitors of PR3 (elafin) or CG/NE (SLP1) or Abs against NB1 (MEM166) or CD18. 
  • Primary isolated HUVECs were used between passages 3 and 4 and pre-stimulated with IL-1β before culturing in Vena8 Endothelial+ biochips using the Kima pump; standard protocol
  • Before perfusion, HUVECs were treated with blocking Abs specific for heterophilic NB1 binding region of PECAM-1. 
  • Perfusion of neutrophils over HUVECs in the biochips was executed via Mirus Evo Nanopump for 8-assays in parallel or the ExiGo pump for single assays at a shear stress of 1 dyne/cm2.
  • Results:  Disrupting NB1-PECAM-1 interactions with mAb PECAM-1 did not disrupt neutrophil adhesion to the endothelial surface.  In contrast, both NB1 and neutrophil serine protease activity are required for neutrophil transmigration.

 

 

 


 Melanoma:  Metastatic movement of melanoma cells in VenaT4 biochip under flow conditions.

  • Microchannels of the VenaT4 biochip were coated with rhVCAM-1 via standard pipette.  Microwells of the biochip were filled with gel containing chemoattractant (fibronectin 5μg/mL)
  • Melanoma cell line 1205-Lu were perfused through the channels using either Mirus Evo Nanopump or the ExiGo pump at a shear flow rate of 0.5 dyne/cm2.
  • Results: 1205-Lu adhered to rhVCAM under shear stress and transmigrated through the 8μm diameter pore size of the VenaT4 biochip invading the underlying microwell.  It was shown that the rate of transmigration in the control microwell was low compared to the transmigration of cells into the microwell containing the chemoattractant.