Non-invasive in vivo monitoring of transplanted stem cells in 3D-bioprinted constructs using near-infrared fluorescent imaging

Abstract

Cell-based tissue engineering strategies have been widely established. However, the contributions of the transplanted cells within the tissue-engineered scaffolds to the process of tissue regeneration remain poorly understood. Near-infrared (NIR) fluorescence imaging systems have great potential to non-invasively monitor the transplanted cell-based tissue constructs. In this study, labeling mesenchymal stem cells (MSCs) using a lipophilic pentamethine indocyanine (CTNF127, emission at 700 nm) as a NIR fluorophore was optimized, and the CTNF127-labeled MSCs (NIR-MSCs) were printed embedding in gelatin methacryloyl bioink. The NIR-MSCs-loaded bioink showed excellent printability. In addition, NIR-MSCs in the 3D constructs showed high cell viability and signal stability for an extended period in vitro. Finally, we were able to non-invasively monitor the NIR-MSCs in constructs after implantation in a rat calvarial bone defect model, and the transplanted cells contributed to tissue formation without specific staining. This NIR-based imaging system for non-invasive cell monitoring in vivo could play an active role in validating the cell fate in cell-based tissue engineering applications.

Keywords: near‐infrared fluorescence; non‐invasive monitoring; scaffold monitoring; stem cell tracking.

FIGURE 1

Schematic of the non‐invasive near‐infrared (NIR) imaging system used to track stem cells (a) Bioink prepared by mixing GelMA‐based materials (including other excipients) and NIR‐mesenchymal stem cells (MSCs). (i) GelMA synthesis, (ii) NIR‐MSCs preparation. NIR‐MSCs: Fluorescence‐emitting (700 nm) CTNF127‐labeled placenta‐derived mesenchymal stem cells (PMSCs) (b) 3D bioprinting of hybrid constructs; PCLG‐copolymer for a framework and NIR‐MSCs‐GelMA for cells were printed alternately. (c) Transplantation into rat calvarial defect and non‐invasive stem cell imaging

FIGURE 2

Optimization of labeling conditions (a, b) cytocompatibility using live/dead assay and fluorescence emission depending on CTNF127 (2 μM) labeling time at day 3 post‐cultivation and their quantification. Scale bars = 250 μm, (c, d) Stability and reliability of near‐infrared (NIR) fluorescence signal intensity as a function of fluorophore concentration (0–8 μM) and culture time (for 7 days), and their quantification. Scale bars = 1.5 cm, ns: not significant (p < 0.05 vs. D3). (e) Microscopic observation at day 20 post‐cultivation (exposure time: 2 s). Scale bars = 250 μm

FIGURE 3

Optimization of printing condition using bioink, near‐infrared‐mesenchymal stem cells‐gelatin methacryloyl (NIR‐MSCs‐GelMA) (a) Single bioink printing. (i–ii) Fluorescence signal intensity from hydrogel printed by bioink with various cell numbers (1–30 M). Every strand in the same construct has different layer numbers (1–6). M: 1 × 10⁶ cells/ml. Fluorescence signal intensity was quantitatively increased by both the printing layer and cell number. The mean fluorescence was normalized by the fluorescence strength of GelMA containing unlabeled PMSCs. (iii) The bioink, composed of 10 M of NIR‐MSCs, was printed in 10 layers, and its imaging reveals sophisticated printing results that show the printed traces in macro‐fluoroscopic measurement. (b) Hybrid 3D bioprinting of bioink and PCLG‐copolymer (i) bioink with various cell numbers every strand (1–30 M) was used to print the alternate lines along PCLG‐copolymer, and their imaging was performed in a one‐layer structure. Bioink demonstrated a reliable increment of fluorescence intensity by cell number. The redder the color, the stronger the intensity in the rainbow image. (ii) Hybrid 3D bioprinting of bioink and PCLG‐copolymer with a lattice structure. NIR fluorescence (700 nm) was distinctly distinguished from the framework of construct regardless of layer number and culture time. Scale bars = 1 cm

FIGURE 4

Long‐term cell viability and fluorescence maintenance in the complex. (a) Representative images from three culture time. Near‐infrared‐mesenchymal stem cells (NIR‐MSCs) shows high cell viability through Dapi/EthD‐1 staining and stable NIR fluorescence emission in long‐term culture (35 days) of the 3D printed complex. Scale bars = 1 mm for phase images, 250 μm for the rest of the images. P: PCLG‐copolymer, N: NIR‐MSCs‐GelMA printed area. Dapi: nuclei, EthD‐1: dead cells (b) Quantitative analysis of (a). (i) The overall number of cells increased in vitro. (ii) The number of living cells dominated the number of dead cells in the GelMA. The entire (iii) signal intensity and (iv) the signal area were not significantly different for 35 days. This reflects that the system composed of CTNF127‐labeled cells embedded in GelMA has high cell viability and low photobleaching and fluorescent efflux. *p < 0.05 versus day 3, ns: not significant

FIGURE 5

Non‐invasive in vivo stem cell monitoring. (a) Implantation of the construct into the rat calvarial defect site and the non‐invasive monitoring for 8 weeks. (i) Observation of experimental group implanted the construct with near‐infrared‐mesenchymal stem cells (NIR‐MSCs). NIR fluorescence was emitted from the implants at 700 nm. (The fluorescence at 4 weeks came from the unshaved fine hairs.) *Control group: the group implanted the constructs without NIR‐MSCs (see Figure S2 for extended analysis). Scale bars = 1 cm. Scale bars = 1 cm. (b) Quantitative analysis of (a) (i) SBR (signal to background) remained high and constant during monitoring. There was no significant difference between SBR immediately after implantation and each detection time (p < 0.05). (ii) Comparison of SBR via non‐invasive imaging (closed skin) and invasive imaging (open skin) at 8 weeks post‐surgery. The SBRs between them were not significantly different (p < 0.05)

FIGURE 6

Stem cell tracking via comparison of near‐infrared (NIR) fluorescence and histological analysis of constructs implanted in calvarial defects for 8 weeks (a) total implanted area. Dotted lines mark the boundary of implant and host tissue. (b) Magnified images of dotted box (denoted as #) in (a). The dotted circle refers to the PCLG‐copolymer (denoted as P), and the surrounding area is the area printed with NIR‐MSCs‐GelMA. The red color indicates fluorescence from NIR‐MSCs (marked as N) and blue color from cells' nuclei within the area. H indicates host tissue. NIR‐MSCs infiltrated into PCLG‐copolymer were marked with an asterisk (*). It was found that the newly formed tissue area (denoted as T) on the HE stained slide overlaps the NIR‐MSCs region. Scale bars = 2.5 mm for (a) and 500 μm for (b)