Sp5 x mp

Structural analysis of the macroporous gel-vaccine was performed using a LEO 982 scanning electron microscope (SEM) (LEO Electron Microscopy). To prepare the samples, cryogels in the frozen state following cryogelation were lyophilized and sectioned for observation. The average size of pores in cryogels was calculated by averaging the diameters of the pores in the gels observed by SEM. The distribution of cells within the scaffolds was visualized with an inverted laser scanning confocal microscope (Leica SP5 XMP, Germany). High-resolution image stacks were collected with 300-nm separation between slices (z-stacks) for the 3D reconstruction of the entire scaffold and visualization of cell-matrix interactions.

Cell proliferation within 3D hydrogels (n=4) at days 1, 4 and 7were assessed qualitatively and quantitatively using Click-iT Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo Fisher Scientific). Cell-laden hydrogels were incubated with 10 μM EdU at 37°C for 24 hours. The samples were then fixed with 4% paraformaldehyde (PFA) for 30 min and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 40 min. Click-iT Plus reaction cocktail was added into the samples and incubated for 40 min in the dark. After another rinse with 3% bovine serum albumin (BSA), the samples were stained using Hoechst 33342 (1:2000) at room temperature. CFs cultured on 2D well plates (5000 cells/well, n=4) were taken as control. A confocal microscope (Leica SP5 X MP, Germany) was used to take fluorescence images of the samples. Acquisition parameters were not changed throughout the imaging of each experiment. The amount of EdU positive cells and Hoechst positive cells were analyzed using the ImageJ program, respectively. The value obtained from the EdU positive cells divided by the total number of Hoechst positive cells was determined to be the percentage of cell proliferation.

All bright field and fluorescent images were taken on a Zeiss Axio Observer D1 Fluorescence Microscope (Carl Zeiss, Germany) and all scanning electron microscopy images were taken on a Hitachi Model S4700, Japan. Confocal images were taken on Leica SP5X MP, Germany.

Samples were fixed for 1 h at room temperature using 4 vol% paraformaldehyde (Sigma-Aldrich) in PBS. Cells were permeabilized by soaking the samples in 0.1 vol% Triton X-100 (Sigma-Aldrich) dissolved in PBS for 30 min while non-specific binding was inhibited using 10 vol% bovine serum albumin (BSA, Sigma-Aldrich) for 1 h at room temperature. Samples were then incubated for overnight at 4 °C in a solution containing primary antibodies at 1:200 dilution in 10 vol% BSA and 0.1 vol% Triton X-100 in PBS. In particular, rabbit polyclonal anti-CD31 (ab32457, Abcam), mouse monoclonal anti-sarcomeric α-actinin (ab9465, Abcam), and rabbit polyclonal anti-connecxin-43 (ab11370, Abcam) antibodies were used. Secondary antibodies were used at 1:200 dilution. For F-actin staining, samples were incubated for 30 min at room temperature in a solution of Alexa 488-phalloidin (A12379, ThermoFisher) at 1:40 dilution in 10 vol% BSA and 0.1 vol% Triton X-100 in PBS. Nuclei of the cells were stained by 4′,6-diamidino-2-phenylindole (DAPI, ThermoFisher). Images were taken using a fluorescence optical microscope (Axio Observer D1, Zeiss) or a confocal fluorescence microscope (SP5 X MP, Leica).

First, 1.4 × 10⁶ GFP-expressing HUVECs were encapsulated in the gelatin-IOHs prepared using alginate beads with 800 μm diameter according to above procedure. After culturing the resulting gelatin-IOHs for 3 d, 1.4 × 10⁶ mCherry-expressing MSCs were seeded on the IOH gels and the cells were cultured for 3 d. The IOHs were fixed in 4% paraformaldehyde and imaged on the confocal microscope (Leica SP5 XMP).

Confocal reflectance microscopy was used to determine the degree of collagen fiber alignment. At least four samples of each condition were imaged on a Leica SP5 X MP confocal/multiphoton microscope system with a 40X (NA = 1.25) oil immersion objective. Images with significant tearing, due to the needle tip dragging when it was cut were removed from analysis. In addition, images were discarded if the ripped area was larger than 25% of the total image area due to needle rotation. The images were then averaged using a Gaussian blur filter with a standard deviation of 2 and analyzed with the ImageJ plugin OrientationJ [31 ] with a cubic spline gradient and a Gaussian window of 4 pixels. The coherency and orientation images were then saved and used later in the analysis. A Matlab (Mathworks, Natick, MA, USA) script was written which identifies average collagen fiber orientation and builds a circumferential vector field. The coherency and orientation images were then used to compare the fiber orientation to a circumferential vector field using coherency cutoff of 0.2. The angle between the local collagen fiber orientation vector and a circumferential vector field is θ_f. The directionality of the fiber field was defined in the following way.
$$D{I}_f=\text{cos}{\theta }_f$$
When the fiber orientation is circumferential, the DI_f is 1 and when the fiber orientation is radial, the DI_f is 0.