In vivo bioluminescent tracking of mesenchymal stem cells within large hydrogel constructs

Abstract

The use of multicomponent scaffolds for cell implantation has necessitated sophisticated techniques for tracking of cell survival in vivo. Bioluminescent imaging (BLI) has emerged as a noninvasive tool for evaluating the therapeutic potential of cell-based tissue engineering strategies. However, the ability to use BLI measurements to longitudinally assess large 3D cellular constructs in vivo and the effects of potential confounding factors are poorly understood. In this study, luciferase-expressing human mesenchymal stem cells (hMSCs) were delivered subcutaneously within agarose and RGD-functionalized alginate hydrogel vehicles to investigate the impact of construct composition and tissue formation on BLI signal. Results showed that alginate constructs exhibited twofold greater BLI counts than agarose constructs at comparable hMSC doses. However, each hydrogel type produced a linear correlation between BLI counts and live cell number, indicating that within a given material, relative differences in cell number could be accurately assessed at early time points. The survival efficiency of delivered hMSCs was highest for the lower cell doses embedded within alginate matrix. BLI signal remained predictive of live cell number through 1 week in vivo, although the strength of correlation decreased over time. Irrespective of hydrogel type or initial hMSC seeding dose, all constructs demonstrated a degree of vascularization and development of a fibrotic capsule after 1 week. Formation of tissue within and adjacent to the constructs was accompanied by an attenuation of BLI signal during the initial period of the image acquisition time-frame. In alginate constructs only, greater vessel volume led to a delayed rise in BLI signal following luciferin delivery. This study identified vascular and fibrotic tissue ingrowth as potential confounding variables for longitudinal BLI studies. Further investigation into the complexities of noninvasive BLI data acquisition from multicomponent constructs, following implantation and subsequent tissue formation, is warranted.

FIG. 1.

Characterization of green fluorescent protein (GFP)/luciferase (Luc)-labeled human mesenchymal stem cells (hMSCs). (A) Labeled hMSCs retained a fusiform morphology and expressed GFP (green) as early as 24 h following lentiviral cotransduction (scale bar=100 μm). (B) Flow cytometry analysis of GFP expression showed 91.9% cells to be labeled. (C) hMSC proliferation was not affected by GFP/Luc labeling. (D) Frequency of luciferin exposure had no impact on hMSC viability over 48 h in culture. Color images available online at www.liebertpub.com/tec

FIG. 2.

Evaluation of dual-syringe hydrogel/cell delivery. Labeled-hMSCs were incorporated into 2% w/v hydrogels using a dual-syringe technique. (A) Seeded hMSCs remained largely viable (live=GFP [green]; dead=ethidium homodimer [red]), retained GFP expression, and were well distributed following embedding (scale bar=500 μm). (B) Metabolic assays showed strong positive correlation to the seeding density of hMSC-seeded agarose and alginate hydrogels. Color images available online at www.liebertpub.com/tec

FIG. 3.

Development of bioluminescent imaging (BLI) protocol and determination of hMSC survival. Agarose and RGD-alginate constructs were used to deliver a range of hMSC doses: 0–2.0×10⁶. Positive correlation between BLI signal and implanted cell number was observed at all imaging acquisition time points (10, 20, and 30 min) following luciferin delivery. (A) Representative BLI heat-maps for agarose and alginate constructs using the 30-min BLI protocol are shown. (B) For agarose hydrogels, the highest correlation coefficient (r²=0.706) was observed using the 30-min BLI protocol. (C) The best fit (r²=0.912) for alginate hydrogels was also achieved using the 30-min BLI protocol. (D) By day 7 postimplantation, the number of live cells had decreased in all agarose cell groups, with the 0.25×10⁶ dose exhibiting proportionately the greatest cell survival. (E) Within the alginate delivery system, the higher cell groups showed proportionately smaller live cell numbers after 1 week. The 0.25×10⁶ cell dose group had improved hMSC survival in comparison to the 2.0×10⁶ dose (^$p<0.05 as indicated; *p<0.05 compared to day 0). Color images available online at www.liebertpub.com/tec

FIG. 4.

Shift in BLI profile after 1 week in vivo. (A) At day 7, construct BLI was positively correlated to GFP⁺ events (r²=0.422). (B) BLI signal exhibited a delayed increase to 30-min value after 1 week in vivo (solid line=day 0 average; dashed line=day 7 average). Day-0 and day-7 cell dose averages represented by solid and outlined symbols, respectively. (C) For agarose constructs, the increase in BLI signal following luciferin injection was attenuated on day 7 compared with day 0. (D) Alginate constructs also showed a slower rate of progression to the 30-min BLI signal on day 7. Color images available online at www.liebertpub.com/tec

FIG. 5.

Construct encapsulation and tissue ingrowth at 1 week postimplantation in vivo. (A) Construct interiors were predominantly comprised of hydrogel islands (H) and the nanofiber mesh (M) exterior was lined with cells (red), fibrous capsule (blue), and regions of vascularized tissue (perfusion agent=black, indicated by yellow arrows; scale bar=100 μm). (B) Hydrogel islands within the construct interior contained vacant and cell-occupied pockets. Alginate constructs of acellular (above) and 2×10⁶ cell dose (below) are shown (scale bar=500 μm). (C) Tissue ingrowth through macroscopic pores (P; labeled in upper image only) in the construct mesh was variable (scale bar=500 μm). (D) Fibrous capsule extended along the mesh exterior and across the tube end. Construct ends contained mixed degrees of cellular infiltration and vascularization (scale bar=500 μm). Color images available online at www.liebertpub.com/tec

FIG. 6.

Construct vascularization at 1 week postimplantation in vivo. (A) Blood vessels were present along the construct exterior (orientation as indicated by illustration) as well as infiltrating macropores of the nanofiber mesh tube (axial view; dashed line signifies location of vascular contouring). (B) Agarose construct vascular volume was independent of delivered hMSC dose. (C) hMSC dose did not impact vasculature formed within alginate constructs. (D) Vasculature (indicated by yellow arrows) within agarose and alginate constructs was confirmed histologically by brightfield microscopy (perfusion agent=black) and von Willebrand factor (vWF) immunostaining (endothelium=red, cell nuclei=blue; scale bar=100 μm). Color images available online at www.liebertpub.com/tec

FIG. 7.

Construct vascularization and BLI signal profile with hydrogel type. (A) Thirty-minute BLI signal and vasculature for hMSC-seeded agarose constructs were not correlated. (B) Construct vasculature did not impact the BLI signal profile for agarose delivery vehicles on day 7. (C) Thirty-minute BLI signal and vasculature for alginate constructs were not correlated. (D) Vascular volume perturbed the BLI signal profile for alginate constructs on day 7. Increased vasculature was negatively correlated with an increase in BLI signal over the initial 10 min following luciferin injection and positively correlated with the rise in BLI signal thereafter.