The hypothesis that scaffold types affect cell morphology and influence cell behavior by altering organelle structure and function has been investigated by acquiring confocal laser scanning microscopy images of cells (z-stacks) on 10 different scaffolds. The primary human bone marrow stromal cells (hBMSCs) were cultured in biomaterial scaffolds such as Spun Coat Polymer Films (SC), Spun Coat Polymer Films with Osteogenic Supplements (SC+OS), Electrospun Polymeric Nanofibers (NF), Electrospun Polymeric Nanofiber with Osteogenic Supplements (NF+OS), Electrospun Polymeric Microfibers (MF), Porous Polysterene Scaffolds (PPS), Matrigel (MG), Fibrin Gel (FG), Collagen Gel (CG), and Collagen Fibrils (CF). More than 100 cells were imaged per scaffold type to provide statistical confidence in the results used for testing the above hypothesis. Images were acquired using a confocal laser scanning microscope (Leica SP5 II, Leica Microsystems). Each cell was stained for actin (Alexa Fluor 546-phalloidin) and nucleus (DAPI), and yielded two z-stacks of image data. Each z-stack consisted of 1024 x 1024 pixels in X-Y dimensions at spatial resolution 0.24 microns per pixel. The number of z-frames per z-stack varied from 12 to 298 (~ 0.71 microns per z frame) as the cell imaging along the Z dimension was attempted to include only contiguous 2D cross sections that included a part of a cell.
The hypothesis that scaffold geometry and material properties affect cell morphology and influence cell behavior by altering organelle structure and function has been investigated by acquiring 3D confocal laser scanning microscopy images of cells (z-stacks) on 3 different polymer (or PLGA) scaffolds. The primary human bone marrow stromal cells (hBMSCs) were cultured in biomaterial scaffolds including Spun Coat Films (SC), Electrospun Microfibers (MF) with the fiber diameter equal to 2.6 µm, and Electrospun Medium Microfibers (MMF) with the fiber diameter equal to 1.1 µm. More than 100 cells were imaged per scaffold type to provide statistical confidence in the results used for testing our hypothesis. Images were acquired using a confocal laser scanning microscope (Leica SP5 II, Leica Microsystems). Each cell was stained with OregonGreen Maleimide (cell membrane stain). The scaffolds (SC, MF and MMF) were made with poly(lactic-co-glycolic acid) (PLGA) conjugated with Flammafluor 648. The imaging yielded two z-stacks of image data per sample (one z-stack for cell, one z-stack for scaffold). Each z-stack consisted of 2048 x 2048 pixels in X-Y dimensions at spatial resolution 0.12 microns per pixel. The number of z-frames per z-stack ranged from 25 (lowest number) to 175 per z-stack (~ 0.462 microns z resolution) as the cell imaging in the Z dimension included only contiguous 2D cross sections that included a part of a cell.
In order to assess the performance of the confocal imaging and image analysis pipeline, fluorescent microspheres with known diameter distributions were imaged and analyzed. Microspheres labeled with fluors corresponding to the two channels used for cell imaging were imaged: FluoSpheres polystyrene microspheres, blue (emission 365 nm, excitation 415 nm, cat. # F-8837), 14.6 ± 0.146 μm diameter, and orange (emission 540 nm, excitation 560 nm, cat. #F-8841), 14.8 ± 0.061 μm diameter (Life Technologies). Five microspheres were imaged for both the actin and nucleus channels using the same confocal microscope settings and voxel dimensions as were used for imaging cells as follows. Microspheres were immersed in DPBS (Dulbecco’s phosphate buffered saline) and imaged with a CLSM (SP5 II confocal microscope, Leica Microsystems) using a 63× water immersion objective (0.9 numerical aperture). Z stacks of images (voxel dimensions of 240 nm × 240 nm × 710 nm, 1 Airy unit, line average 3, 400 Hz) were captured for blue (excitation 405 nm, emission range 434 to 517 nm) and orange (excitation 543 nm, emission range 564 to 663 nm) microspheres.