Measuring the 3D Shape of Organelles in Bone Marrow Stromal Cells During Culture on Nanofiber Scaffolds
Wojtek J. Tutak, Carl G. Simon Jr., Jyotsnendu J. Giri, Peter Bajcsy
Recent work has shown that osteoprogenitor cell culture on nanofiber scaffolds can promote differentiation. This response may be driven by changes in cell morphology caused by the 3D structure of nanofibers. Herein, we hypothesized that nanofiber effects on cell behavior may be mediated by changes in cell organelle structure and function. In order to test this hypothesis, primary human bone marrow stromal cells (hBMSCs) were cultured on poly(- caprolactone) (PCL) nanofiber scaffolds and on flat PCL spuncoat films. After 1 d of culture, hBMSCs were quadruple stained for actin, nucleus, mitochondria and peroxisomes, and then imaged in 3D (three-dimensions) using confocal microscopy. Image analysis revealed that the hBMSC cell body (actin) and peroxisomal volume were reduced during culture on nanofibers. In addition, the nucleus and peroxisomes occupied a larger fraction of the cell volume during culture on nanofibers, suggesting enhancement of the nuclear and peroxisomal functional capacity. Z-Depth measurements indicated that all organelles took on morphologies with greater 3D character on nanofibers. Comparisons of organelle positions indicated that the nucleus, mitochondria and peroxisomes were relatively closer to the cell center (actin) for nanofibers, suggesting that nanofiber culture induced active mechanisms for organelle positioning. The smaller cell volume and more centralized organelle positioning would reduce the energy cost of inter-organelle vesicular transport during culture on nanofibers. Finally, measurements of hBMSC metabolism (DNA, peroxidase, bioreductive potential, lactate and ATP) indicated that peroxidase activity may be enhanced in hBMSCs cultured on nanofibers. These results demonstrate that culture of hBMSCs on nanofibers caused changes in organelle structure, positioning and function, which may affect organelle functional capacity and transport.