Cell rolling, tethering along the microcapillary wall, attachment, firm adhesion and subsequent migration are all key steps in cell-cell signaling and involved in inflammation and therapeutic areas such as oncology, cardiovascular diseases (e.g. atherosclerosis), infectious diseases (e.g. malaria) and respiratory diseases (e.g. asthma and COPD). 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 Selectins, adhesion molecules such as VCAM, ICAM, MadCAM; chemoattractants such as MCP-1, Fractalkine or other proteins of interest. It is also possible to culture endothelial cells (e.g. HUVECs, HMVECs, HLMECs) via the Kima pump, resulting in a set-up for studying cell–cell interaction.
- cell rolling, adhesion & migration assays
- cell observation with brightfield, phase contrast and immunostaining microscopy (all biochips)
- cell observation with confocal microscopy (Vena8 Fluoro+ and Vena8 Glass Coverslip biochips only)
- T-cell, B-cell, platelet, neutrophil, PBMC, monocyte, eosinophil, ligand-interaction studies
- T-cell-endothelial; neutrophil-endothelial; PBMC-endothelial; monocyte-endothelial; eosinophil-endothelial cell interaction studies
- investigation of anti-inflammatory therapies
- Inflammation: Leukocyte adhesion - quantifying adhesion, inhibition, stimulation
- Inositol hexakisphosphate kinase 1 regulated neutrophil function in innate immunity by inhibiting phosphatidylinositol-(3,4,5)-triphosphate signaling.
- Del-1, an Endogenous Leukocyte-Endothelial Adhesion Inhibitor, Limits Inflammatory Cell Recruitment.
Inflammation: Leukocyte adhesion - quantifying adhesion, inhibition, stimulation
Aim: To analyze the role of different adhesion molecules and chemokines involved in various stages of inflammation under physiological flow.
THP1s (monocytic cell line); Monocytes and PBMCs (isolated from healthy donors) were left untreated or pretreated with PMA and perfused over VCAM-1 or E-selectin coated Vena8 Fluoro+ biochip. Alternatively, they were perfused over HUVECs which had been cultured in Vena8 Endothelial+ biochips using the Kima pump; standard protocol.
Perfusion was executed via Mirus Evo Nanopump for 8-assays in parallel or to the ExiGo pump for single assays at a shear stress of 0.5dyne/cm2.
HUVECs were isolated from patients and cultured in biochips with the Kima pump. HUVECs were prestimulated with TNFα or preincubated with anti-VCAM-1 mAb, anti-E-selectin mAb; or all Abs (anti-VCAM-1 Ab; anti-ICAM-1; anti-E-selectin).
Monocyte chemoattractant protein (MCP-1) is an essential chemokine involved in monocyte trafficking, together with its receptor CCR2, and is a major chemoattractant for monocytes to inflamed endothelial cells.
Results: Samples treated with EP4 agonist ONO AE1-329 or PGE2 largely prevented thrombus formation; only small aggregates were observed. Effects of EP4 agonist and PGE2 were reversed by the EP4 antagonist ONO AE3-208.
Data show a specific interaction between cell receptors and purified proteins, with a complete inhibition at a concentration of 50μg/m of antibody in the case of E-selectin:
Difference in adhesion molecule expression levels is reflected in a different degree of adhesion of THP1 to HUVECs under flow conditions:
The sum of the individual Abs does not equal the effect of all Abs used together suggesting a compensatory effect when only one ligand is blocked.
MCP-1 had a much stronger effect on PBMCs compared to THP1 cell line. Data support hypothesis of MCP-1 priming PBMCs for increased adhesion to inflamed endothelium.
Inositol hexakisphosphate kinase 1 regulated neutrophil function in innate immunity by inhibiting phosphatidylinositol-(3,4,5)-triphosphate signaling.
Inositol hexakisphophate kinase 1 (InsP6K1) is one of the three mammalian inositol hexakisphophate kinases that convert inositol hexakisphosphate (InsP6) to InsP7
Adhesion assays: Neutrophils from bone-marrow of mice were stimulated with fMLP and perfused through fibronectin-coated Vena8 Fluoro+ biochip at 10 dyne/cm2.
Detachment assays: fMLP-treated neutrophils were allowed to adhere for 10 mins in fibronectin-coated Vena8 Fluoro+ biochip then increasing shear flow rates were applied at 0.5, 1, 2, 5, 7.5, 10, 12 dyne/cm2 for 1 min per flow rate.
Connect to Mirus Evo Nanopump for 8-assays in parallel or to the ExiGo pump for single assays.
Results: PtdIns(3,4,5)P3 signaling linked to neutrophil trafficking, particularly adhesion and chemotactic migration. Increasing PtdIns(3,4,5)P3 signaling by disruption of InsP6K1 failed to further augment cell adhesion, directionality or migration speed in neutrophils.
Conclusions: Together with in vivo studies, results demonstrate that the attenuated peritoneal accumulation of neutrophils and enhanced bacteria-killing ability of Ip6k1-/- mice were not due to altered neutrophil recruitment.
Del-1, an Endogenous Leukocyte-Endothelial Adhesion Inhibitor, Limits Inflammatory Cell Recruitment.
Endothelially derived secreted molecule, Del-1 (Developmenteal Endothelial Locus-1) is an anti-adhesive factor that interferes with the integrin LFA-1-dependent leukocyte-endothelial adhesion.
Neutrophils from bone-marrow of mice were pre-treated with/without inhibitors and perfused through ICAM-1 or Del-1-coated Vena8 Fluoro+ biochip at 0.8 and 2 dyne/cm2.
Vena8 Fluoro+ biochip were also coated with MIP-2, P-selectin and ICAM-1 or alternatively MIP-2 and P-selectin were co-immobilized with soluble Del-1.
Perfusion: Connect to Mirus Evo Nanopump for 8-assays in parallel or to the ExiGo pump for single assays.
Results: Neutrophils first roll on selectin and then arrest on the integrin ligand. ICAM-1 promotes robust firm adhesion of neutrophils at higher shear rate. Increasing concentrations of Del-1 co-immobilzed with ICAM-1, P-selectin and MIP-2 significantly inhibited neutrophil adhesion to ICAM-1.
Conclusions: Although it is a ligand of LFA-1, Del-1 does not promote firm leukocyte adhesion under flow but interferes with leukocyte adhesion to endothelial ICAM-1.