No abstract

In vitro models of circulatory hemodynamics are required to mimic the microcirculation for study of endothelial cell responses to pulsatile shear stress by live cell imaging. This study reports the design, fabrication and characterisation of a microfluidic device that generates cardiac-like flow in a continuous culture system with a circulatory volume of only 2-3 μL. The device mimics a single chamber heart, with the following cardiac phases: (1) closure of the ventricle inlet valve, (2) contraction of the ventricle (systole) followed by opening of the outlet valve and (3) relaxation of the ventricle (diastole) with opening of the inlet valve whilst the outlet valve remains closed. Periodic valve states and ventricular contractions were actuated by microprocessor controlled pneumatics. The time-dependent velocity-field was characterised by micro-particle image velocimetry (μ-PIV). μ-PIV observations were used to help tune electronic timing of valve states and ventricular contractions for synthesis of an arterial pulse waveform to study the effect of pulsatile shear stress on bovine artery endothelial cells (BAECs). BAECs elongated and aligned with the direction of shear stress after 48 h of exposure to a pulsatile waveform with a maximum shear stress of 0.42 Pa. The threshold for BAECs alignment and elongation under steady (non-pulsatile) flow reported by Kadohama et al. (2006) is 0.7-1.4 Pa. These cells respond to transient shear stress because the time average shear stress of the pulse waveform to generate this morphological response was only 0.09 Pa, well below the steady flow threshold. The microfluidic pulse generator can simulate circulatory hemodynamics for live cell imaging of shear-induced signalling pathways.

SUMMARYThe interstitial and perivascular spaces of the heart contain stem-like cells including cardiac colony forming units -fibroblast (cCFU-F), a sub-fraction of PDGF-AB stimulated S + P + cell proliferation and conversion to myofibroblasts through a metabolic priming effect, yet significantly enhanced anatomical and functional repair via multiple cellular processes. Our study provides a rationale for a novel therapeutic approach to cardiac injury involving stimulating endogenous repair mechanisms via activation of cardiac stem and stromal cells.

Fig. S5 Holotomographic images at single optical levels of a co-culture of fibroblasts cocultured with SAOS-2 showing the presence of branching cell-projections consistent with those inferred from CLSM observations. Page 21: Fig. S6 Sequential holotomographic time-lapse images at a single optical level of a coculture of fibroblasts with SAOS-2, showing rapid movement of a cell projections between timepoints. Several separate cell projections were evident at the interface between two adjacent cells. Page 22: Fig. S7 Flattened optical confocal microscopy projection and two optical levels of fibroblasts co-cultured for 24 h with MM200-B12, showing the presence of organelles derived from different cells on different optical levels. Page 23: Fig. S8 Flattened optical confocal microscopy projections of fibroblasts co-cultured for 24 Hr with MM200-B12, showing tunneling nanotubes. Page 24: Fig. S9 Holotomography demonstrating tunneling nanotubes in a co-culture of fibroblasts with SAOS-2 at a single time point and four separate optical levels. Page 25: Fig. S10 Flattened optical confocal microscopy projection and 6 optical levels of fibroblasts co-cultured with MM200-B12, showing close entwining of fibroblast processes with malignant cells in absence of non-specific transfer of fibroblast label. Page 26: Fig. S11 Scattergrams from computer simulations showing the relationship between stiffness of individual receptor cells and fluorescence acquired from donor cells, where stiffness of the two cell populations is varied, as well as the median CPP volume exchange for each simulation.