Scanning electron microscopy (SEM) preparation and imaging techniques to visualize cell-material interaction on hydrogels and thermoplastic printed scaffold

Abstract text (incl. figure legends and references)
Introduction

Among several imaging methods to study cell-material interaction, Scanning Electron Microscopy (SEM) is capable of resolving both materials and cellular structures down to the nanometer scale. However, Scanning Electron Microscope typically works under high vacuum conditions, which requires special sample preparation. Cells and water containing substrates need to be dehydrated and dried before investigation at room temperature. Dehydration using a row of water/ethanol mixtures with increasing ethanol concentration and drying using hexamethyldisilazane (HMDS) is an established preparation route to visualize cells on vacuum stable substrates by SEM. For water-containing substrates like hydrogels, cryo-SEM or ESEM can be used to study cells material interactions without morphological changes of the substrate due to drying (1).

Here we show different preparation and imaging protocols to visualize with minimalized artifacts resulting from sample preparation, cells" performance (i.e. distribution and morphology) on two types of bio-fabricated substrates commonly used for tissue engineering – hydrogels and thermoplastic (polycaprolactone, PCL) printed scaffolds.

3-dimensional cell culture scaffolds were obtained using 3D printing: hydrogels samples were obtained via extrusion process of alginate-based ink, PCL constructs were prepared using melt electrowriting approach. Mouse fibroblast (NIH3T3) cells were cultured on the scaffolds and visualized using SEM. Cell-seeded PCL scaffolds were investigated by SEM after dehydration and drying using a row of water/ethanol mixtures and HMDS. Hydrogel samples were imaged using cryo-SEM and ESEM following optimized protocols.

SEM of PCL scaffolds allowed detailed observation of cell-material interactions. Focal adhesion points after 3 days of growth were frequently observed with high resolution. Cells grown inside the PCL scaffold can be imaged by embedding the dehydrated and dried sample in frozen ethanol and cutting thin slices by a blade under a stereomicroscope.

Cryo-SEM of hydrogel samples, following the proposed preparation procedure, allowed to study cell attachments without morphological change of substrate and cells. For the investigation of cells and substrates in fully wet/swollen conditions, ESEM was performed at 3°C and 750 Pa water vapor pressure. Due to the limited field of view (diameter of the pressure limiting aperture is 500 µm) stitching of individual images is necessary to visualize the complete scaffold. Using ESEM starting at 3°C and 750 Pa water vapor pressure an in-situ freeze-drying experiment shows the morphological change of gelatin from a dense hydrogel to a porous polymer structure during temperature and pressure decrease according to the pressure-temperature phase diagram of gelatin (see Fig. 1).

SEM is a powerful tool to study cell-material interactions. For water-containing substrates like hydrogels cryo-SEM or ESEM can be used to prevent morphological changes of the substrate due to drying inside vacuum/under atmospheric conditions at room temperature. SEM of freeze-dried samples should be avoided when characterizing hydrogel-based materials in detail.

Fig. 1: Morphological change of aqueous 1.8 wt.% gelatin during in-situ freeze-drying using ESEM.