In Vivo Subretinal ARPE-19 Cell Tracking Using Indocyanine Green Contrast-Enhanced Multimodality Photoacoustic Microscopy, Optical Coherence Tomography, and Fluorescence Imaging for Regenerative Medicine

Abstract

Purpose: Cell-based regenerative therapies are being investigated as a novel treatment method to treat currently incurable eye diseases, such as geographic atrophy in macular degeneration. Photoacoustic imaging is a promising technology which can visualize transplanted stem cells in vivo longitudinally over time in the retina. In this study, a US Food and Drug Administration (FDA)-approved indocyanine green (ICG) contrast agent is used for labeling and tracking cell distribution and viability using multimodal photoacoustic microscopy (PAM), optical coherence tomography (OCT), and fluorescence imaging.

Methods: Twelve rabbits (2.4-3.4 kg weight, 2-4 months old) were used in the study. Human retinal pigment epithelial cells (ARPE-19) were labeled with ICG dye and transplanted in the subretinal space in the rabbits. Longitudinal PAM, OCT, and fluorescence imaging was performed for up to 28 days following subretinal administration of ARPE-19 cells.

Results: Cell migration location, viability, and cell layer thickness were clearly recognized and determined from the fluorescence, OCT, and PAM signal. The in vivo results demonstrated that fluorescence signal increased 37-fold and PAM signal enhanced 20-fold post transplantation.

Conclusions: This study demonstrates that ICG-assisted PAM, OCT, and fluorescence imaging can provide a unique platform for tracking ARPE-19 cells longitudinally with high resolution and high image contrast.

Translational relevance: Multimodal PAM, OCT, and fluorescence in vivo imaging with ICG can improve our understanding of the fate, distribution, and function of regenerative cell therapies over time nondestructively.

Figure 1.

Optical characterization, in vitro toxicity and cellular uptake of ICG. (A) UV-Vis absorption spectrum of ICG dye obtained at various concentrations (i.e. 0 [saline], 6.25, 12.5, 25, 50, and 100 µg/mL). (B) UV-Vis absorption spectrum of ICG labeled ARPE-19 cells measured from the samples incubated with 250 µg/mL at 24 hours, 48 hours, and 72 hours. (C) Absorption spectra of the treated cells under laser illumination at various energies from 0 to 640 nJ, revealing photostability of the labeled cells. (D) Cytotoxicity of ICG in ARPE-19 cells. ICG at different concentrations ranging from 0 to 500 µg/mL was cultured with ARPE-19 cell at various time points: 24 hours and 48 hours. (E) Cell viability of ARPE-19 cells labeled with ICG at concentration of 200 µg/mL followed laser illumination at different laser fluences (i.e. 0.0025, 0.005, 0.01, 0.02, and 0.04 mJ/cm²). (F–H) Flow cytometry analysis obtained from three groups: control group D, treated group with ICG at 250 µg/mL and incubated for 24 hours E, and 48 hours (F), n = 3, P < 0.001. (I–L) Confocal laser scanning macroscopic images of the internalized ICG inside the cells. ARPE-19 cells were cultured with ICG suspension solution at different concentrations (i.e. 0 [control], 150, 200, and 250 µg/mL) for 24 hours. Pseudo-green fluorescence color illustrates the distribution of internalized ICG inside the cells.

Figure 2.

In vitro photoacoustic microscopy of single ARPE-19 cells. (A–D) In vitro PAM images of single ARPE-19 cells. The PAM images were obtained using the excitation wavelength of 700 nm and laser energy of 80 nJ. (E) Graph of fluorescent intensity as a function of concentrations. (F) Quantitative measurement of photoacoustic signal. (G) In vitro biodegradable of ICG dyes in bioenvironment. The fluorescent signal gradually reduced over time (n = 5, P < 0.05).

Figure 3.

Multimodal imaging visualization of ARPE-19 cells in living rabbits. Color fundus photography, ICG, and PAM pre- and post-administration of ICG-RGD at different time points: 0 (immediately after injection), 1, 3, 5, 7, 10, 14, 21, and day 28. (A, B) Color fundus photography and fluorescence images before and after subretinal injection of ICG labeled-ARPE-19 cells into the subretinal space in the rabbit retina. These images were achieved longitudinally over a period of 28 days. The location of the cells after transplantation was clearly observed with strong fluorescence contrast (red dotted circle). The strongest fluorescence signal occurred from day 0 to day 5 post injection. (D, E) Maximum intensity projection (MIP) PAM images before and after cells transplantation acquired from two different wavelength of 578 and 700 nm. White arrow indicates the position of the transplanted cells. (E) Overlay PAM images acquired at 578 and 700 nm on the same imaging planes. Pseudo-cyan fluorescence color represents the cell viability as well as the migration pattern. (F, G) Quantitative measurement fluorescence signal intensity and PAM signal amplitudes, respectively.

Figure 4.

In vivo evaluation of subretinal injection of ICG suspension solution. Dynamic change of ICG in the subretinal space after subretinal injection was examined longitudinally. (A, B) color and fluorescence photographs of the rabbit retina before and after subretinal injection of ICG (20 µg/mL, 30 µL). Biodistribution of ICG in the subretinal space was clearly observed with strong fluorescent signal immediately after the injection (red dotted circle) and at day 1 post-injection. (C, D) Maximum intensity projection (MIP) PAM images obtained at 578 and 700 nm, respectively. White arrows indicate the distribution of ICG. The strongest PA signal was visible on the PAM acquired immediately post-injection. Then, the PAM signal rapidly reduced and was completely gone by day 7 post-injection.

Figure 5.

In vivo visualization of dead ARPE-19 cells labeled with ICG. (A, B) Color fundus and fluorescence photographs of the rabbit retina pre– and post-subretinal injection of 30 µL dead ICG labeled ARPE-19 cells at a density of 1 × 10⁶ cells per millimeter. (C, D) MIP PAM images acquired at the excitation wavelengths of 578 and 700 nm, respectively. Dead cells were observed on the PAM image obtained at 700 nm and fluorescence images immediately and at day 1 post-injection (white arrows). Rapid reduction in the fluorescence and PAM signal was noted with the dead cells by 5 days with minimal signal by day 7 post-injection.

Figure 6.

In vivo photostability analysis and spectroscopic PAM image. (A) MIP PAM images acquired at 578 nm and 700 nm at the same scanning areas. Noted that the PAM images at 700 nm were repeated three times. White arrows show the detected ARPE-19 cells. (B) A graph of PAM signal amplitudes isolated at the position of the detected cells. The quantitative PAM amplitudes were not significantly different among three scans and exhibited 4% fluctuation (i.e. 3.68 ± 0.43 a.u. for the first scan, and 3.53 ± 0.31 a.u. for the third scan, P = 0.25 > 0.05). (C) spectroscopic PAM images acquired at different wavelengths ranging from 563 nm to 700 nm. (D) The 3D rendering visualization of the PAM data.

Figure 7.

Optical coherence tomography (OCT) image of ARPE-19 cells. (A–J) Longitudinal B-scan OCT images obtained before and after cell transplantation. OCT image before the injection shows different intact retinal layers A, whereas localized retinal detachment was observed on the OCT image after injection B. Note that labeled cells were detected (white arrows) and randomly distributed in the subretinal space along with the accumulation of subretinal fluid. (K) The 3D OCT visualization. Encoded color shows different depths of retinal layers.

Figure 8.

Histopathological and immunohistochemistry analysis. Hematoxylin and eosin (H&E) images of control group (A) and cell transplantation group (B). The transplanted ARPE-19 were clearly visualized in the subretinal space (yellow arrow). (C–L) immunofluorescence images of the control and cell transplanted tissues. Red fluorescence color indicates the RPE-65 positive. Blue color stained by DAPI. Brightfield images show the structure of retinal tissues. Images show the location ARPE-19 cells (yellow arrows) and native RPE cells (white arrows), which is consistent with OCT images.