Tom J. A. Kokhuis

Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.

Brandaris 128 ultra-high-speed imaging facility: 10 years of operation, updates, and enhanced features

The Brandaris 128 ultra-high-speed imaging facility has been updated over the last 10 years through modifications made to the cameras hardware and software. At its introduction the camera was able to record 6 sequences of 128 images (500 × 292 pixels) at a maximum frame rate of 25 Mfps. The segmented mode of the camera was revised to allow for subdivision of the 128 image sensors into arbitrary segments (1-128) with an inter-segment time of 17 μs. Furthermore, a region of interest can be selected to increase the number of recordings within a single run of the camera from 6 up to 125. By extending the imaging system with a laser-induced fluorescence setup, time-resolved ultra-high-speed fluorescence imaging of microscopic objects has been enabled. Minor updates to the system are also reported here.

Intravital microscopy of localized stem cell delivery using microbubbles and acoustic radiation force

The use of stem cells for the repair of damaged cardiac tissue after a myocardial infarction holds great promise. However, a common finding in experimental studies is the low number of cells delivered at the area at risk. To improve the delivery, we are currently investigating a novel delivery platform in which stem cells are conjugated with targeted microbubbles, creating echogenic complexes dubbed StemBells. These StemBells vibrate in response to incoming ultrasound waves making them susceptible to acoustic radiation force. The acoustic force can then be employed to propel circulating StemBells from the centerline of the vessel to the wall, facilitating localized stem cell delivery. In this study, we investigate the feasibility of manipulating StemBells acoustically in vivo after injection using a chicken embryo model. Bare stem cells or unsaturated stem cells (<5 bubbles/cell) do not respond to ultrasound application (1 MHz, peak negative acoustical pressure P_ = 200 kPa, 10% duty cycle). However, stem cells which are fully saturated with targeted microbubbles (>30 bubbles/cell) can be propelled toward and arrested at the vessel wall. The mean translational velocities measured are 61 and 177 μm/s for P‐ = 200 and 450 kPa, respectively. This technique therefore offers potential for enhanced and well‐controlled stem cell delivery for improved cardiac repair after a myocardial infarction. Biotechnol. Bioeng. 2015;112: 220–227.

Secondary Bjerknes Forces Deform Targeted Microbubbles

Secondary Bjerknes forces can rupture the binding of targeted microbubbles. We have shown before that this effect can be used to quantify the adhesion strength between bubble and target surface [1]. At lower pressures however, microbubbles were observed to snap back to their original position within 100 µs after ultrasound application. In this study the mechanism of this restoring force was investigated in more detail using simultaneous top and side view high speed imaging [2]. Moreover, some results on the process of microbubble detachment (peeling versus uniform rupture) are presented.

Viability of endothelial cells after ultrasound-mediated sonoporation: Influence of targeting, oscillation, and displacement of microbubbles

Microbubbles (MBs) have been shown to create transient or lethal pores in cell membranes under the influence of ultrasound, known as ultrasound-mediated sonoporation. Several studies have reported enhanced drug delivery or local cell death induced by MBs that are either targeted to a specific biomarker (targeted microbubbles, tMBs) or that are not targeted (non-targeted microbubbles, ntMBs). However, both the exact mechanism and the optimal acoustic settings for sonoporation are still unknown. In this study we used real-time uptake patterns of propidium iodide, a fluorescent cell impermeable model drug, as a measure for sonoporation. Combined with high-speed optical recordings of MB displacement and ultra-high-speed recordings of MB oscillation, we aimed to identify differences in MB behavior responsible for either viable sonoporation or cell death. We compared ntMBs and tMBs with identical shell compositions exposed to long acoustic pulses (500-50,000cycles) at various pressures (150-500kPa). Propidium iodide uptake highly correlated with cell viability; when the fluorescence intensity still increased 120s after opening of the pore, this resulted in cell death. Higher acoustic pressures and longer cycles resulted in more displacing MBs and enhanced sonoporation. Non-displacing MBs were found to be the main contributor to cell death, while displacement of tMBs enhanced reversible sonoporation and preserved cell viability. Consequently, each therapeutic application requires different settings: non-displacing ntMBs or tMBs are advantageous for therapies requiring cell death, especially at 500kPa and 50,000cycles, whereas short acoustic pulses causing limited displacement should be used for drug delivery.

Globular adiponectin controls insulin-mediated vasoreactivity in muscle through AMPKα2

Decreased tissue perfusion increases the risk of developing insulin resistance and cardiovascular disease in obesity, and decreased levels of globular adiponectin (gAdn) have been proposed to contribute to this risk. We hypothesized that gAdn controls insulins vasoactive effects through AMP-activated protein kinase (AMPK), specifically its α2 subunit, and studied the mechanisms involved. In healthy volunteers, we found that decreased plasma gAdn levels in obese subjects associate with insulin resistance and reduced capillary perfusion during hyperinsulinemia. In cultured human microvascular endothelial cells (HMEC), gAdn increased AMPK activity. In isolated muscle resistance arteries gAdn uncovered insulin-induced vasodilation by selectively inhibiting insulin-induced activation of ERK1/2, and the AMPK inhibitor compound C as well as genetic deletion of AMPKα2 blunted insulin-induced vasodilation. In HMEC deletion of AMPKα2 abolished insulin-induced Ser(1177) phosphorylation of eNOS. In mice we confirmed that AMPKα2 deficiency decreases insulin sensitivity, and this was accompanied by decreased muscle microvascular blood volume during hyperinsulinemia in vivo. This impairment was accompanied by a decrease in arterial Ser(1177) phosphorylation of eNOS, which closely related to AMPK activity. In conclusion, globular adiponectin controls muscle perfusion during hyperinsulinemia through AMPKα2, which determines the balance between NO and ET-1 activity in muscle resistance arteries. Our findings provide a novel mechanism linking reduced gAdn-AMPK signaling to insulin resistance and impaired organ perfusion.

Lipid distribution and viscosity of coated microbubbles

Ultrasound contrast agents consist of gas-filled coated microbubbles with diameters between 1 and 10 µm. Within an ultrasound field, high differences in responses of similar sized microbubbles have been reported. Heterogeneous coating properties have been suggested to be the underlying cause. Until now, properties of this coating, like viscosity have been studied dynamically using a set-up of vibrating microbubbles in an ultrasound field. This study focuses on determining the viscosity of the coating for lipid-coated microbubbles in a static set-up. The viscosity of the coating was determined by measuring the mobility of a fluorescent lipid using Fluorescence Recovery After Photobleaching (FRAP). We found a surface shear viscosity of 8×10−6 kg/s that was independent of the microbubble size. In addition, we found that the lipid distribution in the coating was heterogeneous and varied from microbubble to microbubble. In conclusion, this study shows that the static surface shear viscosity of the coating can be determined in an independent way which can now be used in microbubble dynamics models.

Segmented high speed imaging of vibrating microbubbles during long ultrasound pulses

Detailed information about the response of microbubbles to long ultrasound pulses (>100 cycles) is hampered by the limited time span ultra fast-framing cameras (> 10 MHz) cover. We therefore developed a new imaging mode for the Brandaris 128 camera [1], facilitating high speed imaging during small time windows (segments), equally distributed over a relatively large time span.

Surface contact of bound targeted microbubbles

For molecular imaging using ultrasound contrast agents, targeted microbubbles are designed with specific ligands linked to the coated shell. Research is ongoing to determine the binding force of targeted microbubbles and to distinguish bound from unbound targeted microbubbles using ultrasound. For this, the actual surface of the targeted microbubbles that binds to a pathology is important. This study focuses on determining the surface contact of bound targeted microbubbles by fluorescence microscopy. Biotinylated lipid-coated microbubbles (3-7 μm in diameter) with either DSPC or DPPC as the main lipid were targeted to a streptavidin-coated surface. The binding area of targeted microbubbles was found to be 6 ± 4% of the total microbubble surface for microbubbles with DSPC as the main lipid (n=22) and 11 ± 4% for microbubbles with DPPC as the main lipid (n=24). The difference can be explained by the heterogeneous distribution of the ligand for DSPC microbubbles whereas the ligand is homogeneously distributed for DPPC microbubbles. These findings can be used to improve the binding of targeted microbubbles and for the ongoing research to distinguish bound from unbound microbubbles.

Secondary Bjerknes forces deform targeted microbubbles

Secondary Bjerknes forces can rupture the binding of targeted microbubbles. We have shown before that this effect can be used to quantify the adhesion strength between bubble and target surface [1]. At lower pressures however, microbubbles were observed to snap back to their original position within 100 µs after ultrasound application. In this study the mechanism of this restoring force was investigated in more detail using simultaneous top and side view high speed imaging [2]. Moreover, some results on the process of microbubble detachment (peeling versus uniform rupture) are presented.