Toxicity and imaging of multi-walled carbon nanotubes in human macrophage cells.

Multi-walled carbon nanotubes (MWNTs) have been proposed for use in many applications and concerns about their potential effect on human health have led to the interest in understanding the interactions between MWNTs and human cells. One important technique is the visualisation of the intracellular distribution of MWNTs. We exposed human macrophage cells to unpurified MWNTs and found that a decrease in cell viability was correlated with uptake of MWNTs due to mainly necrosis. Cells treated with purified MWNTs and the main contaminant Fe(2)O(3) itself yielded toxicity only from the nanotubes and not from the Fe(2)O(3). We used 3-D dark-field scanning transmission electron microscopy (DF-STEM) tomography of freeze-dried whole cells as well as confocal and scanning electron microscopy (SEM) to image the cellular uptake and distribution of unpurified MWNTs. We observed that unpurified MWNTs entered the cell both actively and passively frequently inserting through the plasma membrane into the cytoplasm and the nucleus. These suggest that MWNTs may cause incomplete phagocytosis or mechanically pierce through the plasma membrane and result in oxidative stress and cell death.

Generating suspended cell monolayers for mechanobiological studies.

Cell monolayers line most of the surfaces and cavities in the human body. During development and normal physiology, monolayers sustain, detect and generate mechanical stresses, yet little is known about their mechanical properties. We describe a cell culture and mechanical testing protocol for generating freely suspended cell monolayers and examining their mechanical and biological response to uniaxial stretch. Cells are cultured on temporary collagen scaffolds polymerized between two parallel glass capillaries. Once cells form a monolayer covering the collagen and the capillaries, the scaffold is removed with collagenase, leaving the monolayer suspended between the test rods. The suspended monolayers are subjected to stretching by prying the capillaries apart with a micromanipulator. The applied force can be measured for the characterization of monolayer mechanics. Monolayers can be imaged with standard optical microscopy to examine changes in cell morphology and subcellular organization concomitant with stretch. The entire preparation and testing protocol requires 3-4 d.