Chronic label-free volumetric photoacoustic microscopy of melanoma cells in three-dimensional porous scaffolds

Abstract

Visualizing cells in three-dimensional (3D) scaffolds has been one of the major challenges in tissue engineering. Most current imaging modalities either suffer from poor penetration depth or require exogenous contrast agents. Here, we demonstrate photoacoustic microscopy (PAM) of the spatial distribution and temporal proliferation of cells inside three-dimensional porous scaffolds with thicknesses over 1 mm. Specifically, we evaluated the effects of seeding and culture methods on the spatial distribution of melanoma cells. Spatial distribution of the cells in the scaffold was well-resolved in PAM images. Moreover, the number of cells in the scaffold was quantitatively measured from the as-obtained volumetric information. The cell proliferation profile obtained from PAM correlated well with what was obtained using the traditional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Fig. 1

A schematic diagram of the photoacoustic microscopy system. A Cartesian coordinate is also shown in the drawing.

Fig. 2

SEM images of A) a free-standing, opaline lattice of gelatin microsphere and B) a PLGA inverse opal scaffold. The insets show magnified views of the samples (scale bars: 100 μm).

Fig. 3

A) Optical and B, C) SEM images of a PLGA inverse opal scaffold seeded with melanoma cells acquired at 14 days post-seeding. The images in (B) and (C) show scattered cells and a cluster of cells (indicated by arrows), respectively, inside the pores.

Fig. 4

PA images of melanoma cells in a scaffold acquired at 14 days post-seeding. A) PA coronal (top) and sagittal (side) MAP images. The black dots correspond to melanoma cells. B) 3D depiction of the PA image, where the contour of the scaffold is marked by dotted lines. MAP: maximum amplitude projection.

Fig. 5

PA images (MAP of the layer between 420 and 780 μm from the top of the scaffolds) of a scaffold seeded and cultured continuously in the spinner flask for (A) 1 day and (C) 14 days; and of a scaffold seeded with stationary method and cultured on a tilt-angle shaker for (B) 1 day and (D) 14 days.

Fig. 6

PA MAP images of melanoma cells in a scaffold 14 days post-seeding taken from different layers parallel to the top surface. The first layer started at 420 μm beneath the surface, and the layer spacing was 60 μm. Melanoma cells were seeded and culture with a spinner flask. The cells distributed uniformly in the center of the scaffold.

Fig. 7

A) Time course PA images (coronal MAP of the entire volume) of melanoma cells in a typical scaffold at 1, 3, 7 and 14 days post-seeding. B) Quantitative analysis of melanoma cells in scaffolds derived from both PA imaging (n = 4) and MTT cell viability analysis (n = 3). The data are presented as means ± standard errors. Melanoma cells were seeded with a spinner flask and cultured in still.