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Published in final edited form as: Ultrasound Med Biol. 2020 Sep 26;46(12):3468–3474. doi: 10.1016/j.ultrasmedbio.2020.08.026

A TRIMODAL ULTRASOUND, PHOTOACOUSTIC AND MAGNETIC RESONANCE IMAGING APPROACH FOR LONGITUDINAL POSTOPERATIVE MONITORING OF STEM CELLS IN THE SPINAL CORD

KELSEY P KUBELICK *,†, STANISLAV Y EMELIANOV *,†
PMCID: PMC7709928  NIHMSID: NIHMS1632919  PMID: 32988671

Abstract

Longitudinal monitoring of stem cells in the spinal cord could unveil critical information needed to understand regenerative processes, thereby expediting therapy development and translation. We introduce a post-operative trimodal imaging approach to monitor stem cells in the spinal cord over time. A key aspect of the approach is to label the stem cells with Prussian blue nanocubes (PBNCs), which simultaneously possess optical and magnetic properties for ultrasound-guided photoacoustic (US/PA) and magnetic resonance imaging (MRI) contrast. PBNC-Labeled stem cells were injected into the spinal cord of immunodeficient rats and tracked with US/PA imaging and MRI up to 14 d post-injection. Good agreement was observed between imaging modalities in vivo. Our results suggest that further development of the US/PA/MR imaging approach may create a powerful tool to aid development of regenerative therapies of the spinal cord, and the non-invasive imaging approach can ultimately be deployed in intra- and post-operative environments.

Keywords: Photoacoustic imaging, Magnetic resonance imaging, Ultrasound imaging, Stem cells, Spinal cord, Nanoparticles, Regenerative medicine, Cell tracking

INTRODUCTION

Stem cell therapies have been investigated to treat various conditions of the central nervous system (CNS), including Parkinson’s disease, Alzheimer’s disease, brain tumors, stroke, amyotrophic lateral sclerosis, multiple sclerosis and spinal cord injuries (Lindvall et al. 2004; Lindvall and Kokaia 2010; Donnelly et al. 2012; Gutova et al. 2013). Countless combinations of stem cell types, doses, injection locations and other delivery parameters have been studied with varying degrees of success (Takahashi et al. 2011; Tetzlaff et al. 2011; Lamanna et al. 2017a). Stem cell therapy research would benefit from development of non-invasive imaging tools to guide stem cell delivery and track stem cell location, survival and engraftment over time (Lindvall et al. 2004; Donnelly et al. 2012, 2018; Kubelick and Emelianov 2020a, 2020b). More specifically, longitudinal stem cell monitoring could unveil information enabling us to better understand the regenerative process, standardize therapies and provide information to personalize patient therapy when needed.

In the spinal cord, positron emission tomography, bioluminescence imaging, optical imaging and magnetic resonance imaging (MRI) have all been assessed for stem cell tracking in the spinal cord (Okada et al. 2005; Gutova et al. 2013; Song et al. 2014; Lamanna et al. 2017a). As the gold standard for CNS imaging, MRI approaches are most appealing. Before transplantation, stem cells must be labeled with a contrast agent to generate MR contrast. Superparamagnetic iron oxide nanoparticles (SPIONs) are an excellent choice. Many types are approved or under clinical investigation (Dadfar et al. 2019). For example, ferumoxytol-labeled stem cells could be detected up to 105 d post-injection in porcine models (Lamanna et al. 2017a). However, MRI approaches have several disadvantages including high cost, large footprint and slow image acquisition time. The need for a separate imaging suite also restricts MRI to post-operative, longitudinal monitoring.

Photoacoustic (PA) imaging has been employed in many previous stem cell tracking applications (Jokerst et al. 2012; Nam et al. 2012; Ricles et al. 2014; Kim et al. 2017; Kubelick et al. 2019). As a hybrid optical and acoustic modality, PA imaging allows high-resolution and high-contrast detection at imaging depths greater than those used in purely optical methods (Xu and Wang 2006; Emelianov et al. 2009; Bouchard et al. 2014). Ultrasound-guided photoacoustic (US/PA) imaging has clear advantages over MRI for intra-operative guidance of stem cell therapies in the spinal cord, including real-time imaging, small footprint, portability and operating room compatibility (Donnelly et al. 2018; Kubelick and Emelianov 2020a, 2020b).

To address the need for intra-operative guidance, we recently reported the use of Prussian blue nanocubes (PBNCs) to guide stem cell therapies of the spinal cord with US/PA imaging (Kubelick and Emelianov 2020a, 2020b). PBNCs were selected for stem cell labeling because of their established cytocompatibility (Snider et al. 2018) and synthesis using clinical precursors (Dadfar et al. 2019). Furthermore, optical and magnetic properties of PBNCs generate both PA and MR contrast (Dumani et al. 2020). Our initial in vivo studies illustrated the feasibility of using US/PA imaging for intra-operative guidance and post-operative MR detection 24 h after surgery (Kubelick and Emelianov 2020b). However, use of US/PA imaging does not need to be restricted to the operating room to guide the injection of stem cells. Indeed, use of US/PA imaging can be extended to post-operative monitoring alongside MRI. To this end, the goal of the study described here was to assess the feasibility of using longitudinal, post-operative US/PA and MR imaging to track PBNC-labeled stem cells in the spinal cord. PBNC-Labeled stem cells were non-invasively monitored up to 2 wk after surgery with US/PA imaging, and the results were compared with those for MRI.

METHODS

Nanoparticle synthesis

All chemicals were used as received. Dextran-coated PBNCs with an edge length of approximately 200 nm and a peak absorption of approximately 750-nm wavelength were synthesized in-house using our previously established methods described elsewhere (Snider et al. 2018; Dumani et al. 2020; Kubelick and Emelianov 2020a, 2020b). Briefly, the SPION (10-nm diameter, Ocean Nanotech, San Diego, CA, USA), reactant (5% potassium hexacyanoferrate (II) trihydrate, Sigma-Aldrich, St. Louis, MO, USA) and catalyst (1.85% HCl, Sigma-Aldrich) solutions in deionized ultrafiltered water were combined in that order and stirred for at least 1 h. The solution changed color from light brown to deep blue, indicating formation of PBNCs. PBNCs were dextran coated by adding 10 mg dextran/mg Fe and stirring gently overnight. PBNCs were characterized using transmission electron microscopy (TEM; HT7700, Hitachi, Tokyo, Japan) and UV–Vis spectrophotometry (Evolution 220, ThermoFisher, Waltham, MA, USA). Particles were stored in the dark and were sterilized under UV light for at least 30 min before stem cell labeling.

Stem cell labeling protocol

Human adipose-derived mesenchymal stem cells (MSCs; Lonza, Basel, Switzerland) below passage 8 were cultured according to standard protocols. MSCs were labeled with PBNCs using our previously reported methods described elsewhere (Snider et al. 2018; Kubelick and Emelianov 2020a, 2020b); minimal impact on stem cell viability and multipotency has been observed after labeling (Snider et al. 2018).

In brief, particle-containing medium was prepared by adding PBNCs (∼53 μg Fe/mL medium) to α-minimum essential medium (Corning Life Sciences, Tewksbury, MA, USA) supplemented with 20% fetal bovine serum (Phenix Research, Candler, NC, USA), 5% L-glutamine and 1% penicillin/streptomycin (ThermoFisher). At ∼80% confluence, the standard MSC medium in the tissue culture flask was replaced with PBNC-containing medium. MSCs were incubated with PBNCs at 2 optical density overnight. The next day, PBNC-labeled MSCs were rinsed at least three times with phosphate-buffered saline (PBS) to remove excess particles that were not endocytosed by the MSCs. PBNC-labeled MSCs were collected from the tissue culture flask, suspended at 10,000 cells/μL in phosphate-buffered saline (PBS) and stored on ice before injection.

For histology, PBNC-labeled MSCs were also incubated with ∼30 μM CellTracker Green CMFDA Dye (ThermoFisher) for 45 min. Thus, MSCs were double-labeled with PBNCs and a fluorescent dye before in vivo studies.

Surgical procedure

All studies involving animals were conducted according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Georgia Institute of Technology. The study was conducted in five immunodeficient female nude rats (two 6- to 8-wk-olds weighing 162–176 g and three 39- to 46-wk-olds weighing 210 g). One rat received two injections. Thus, there were a total of six injections. Rats were anesthetized and prepared for surgery by removing hair and sterilizing the exposed skin. Before incision, sustained-release buprenorphine (0.6–0.8 mg/kg) was administered for postoperative analgesia.

Similar to clinical and investigational protocols, a lumbar laminectomy was performed to expose the spinal cord and allow direct injection of PBNC-labeled MSCs into the spinal cord tissue (Boulis et al. 2012; Glass et al. 2012; Feldman et al. 2014; Lamanna et al. 2017b; Donnelly et al. 2018; Kubelick and Emelianov 2020b). Up to 5 μL of PBNC-labeled MSCs suspended at 10,000 cells/μL, a clinically relevant concentration, was injected into the spinal cord in vivo at the rate of 16 nL/s using a 33G syringe attached to an ultra-micropump (World Precision Instruments, Sarasota, FL, USA). The needle remained in the spinal cord for 5 min post-injection to prevent or minimize reflux. Locations of injection sites were recorded. After stem cell injections, the muscle and skin were sutured back over the spinal cord, but per the clinical protocol, the bone was not replaced. After surgery, rats recovered on a heating pad in the surgical suite. All animals regained mobility within several hours. The animals were returned to the housing facility and were monitored to ensure recovery.

Image acquisition

Ultrasound images were acquired with the Vevo 2100 (FUJIFILM VisualSonics, Toronto, ON, Canada) imaging system using a 20-MHz center frequency linear array transducer (LZ250, FUJIFILM VisualSonics). For PA imaging, the Vevo 2100 ultrasound system and LZ250 imaging transducer were interfaced with a laser (Phocus MOBILE, OPOTEK, Carlsbad, CA, USA) operating at 20-Hz pulse repetition frequency, 5- to 7-ns pulse duration and 39 ± 3.5 mJ at 750-nm wavelength. PA images were acquired at 5 frames/s. MR images were acquired using a 7-T pre-clinical system (Bruker PharmaScan, Billerica, MA, USA) and a gradient coil with an inner diameter of 60 mm. The built-in T2-Turbo RARE pulse sequence was used to produce images with T2 contrast, consisting of a repetition time (TR) of 4250 ms, an echo time (TE) of 33 ms, a RARE factor of 8 and two averages.

In vivo US/PA and MR images were acquired at each time point up to 14 d post-injection. Per IACUC guidelines, animals were never imaged on consecutive days, and each imaging session was completed within 4 h. Animals were lightly anesthetized to immobilize for imaging. No animals required early termination from the study; however, modifications to intermediate imaging time points were sometimes required if negative reactions to anesthesia were observed, such as labored breathing and slow heart rate.

For US/PA imaging, rats were secured on a heated platform. The imaging transducer was cleaned with 70% isopropanol, and the wound was cleaned with alcohol swabs (BD, Franklin Lakes, NJ, USA). Kimwipes were taped to the skin on either side of the incision, creating a ∼1-cm window over the spinal cord to reduce background signals outside the region of interest. Three of the immunodeficient rats used in this study had a black stripe on their skin running along the spine. Laser light irradiation of this melanin-rich stripe would reduce fluence at the underlying spinal cord. Given that rats have excess, loose skin, the stripe was kept out of the imaging field of view by moving the skin and temporarily securing it with tape during the imaging experiment. Three-dimensional US/PA data sets were compiled from sequential 2-D axial views acquired with 89–120 μm steps using a translational motor stage. Photoacoustic signals were acquired within the wavelength range 700–950 nm in 50-nm increments.

For MRI, the rat was secured in a cylindrical holder. Sensors were adhered to the skin to monitor breathing and heart rate. The rat was wrapped in a flexible heating pad and inserted in the imaging bore of the MR scanner. T2-Weighted axial images were acquired with a 1-mm slice thickness. The field of view was 5.0–5.5 cm depending on the size of the rat, with an image matrix of 256 × 256 pixels, corresponding to pixel dimensions of ∼0.2 × ∼0.2 mm (width × height). Thus, the voxel size was 0.04 mm3 for axial images.

At the completion of the 14-d-long imaging study, the rats were sacrificed, and the spinal cord was excised for histology. Cross-sectional tissue sections were stained with eosin (VWR International, Radnor, PA, USA) for brightfield microscopy (Axio Observer, Zeiss, Oberkochen, Germany) or 4′,6-diamidino-2-phenylindole (DAPI, ThermoFisher) for fluorescent confocal microscopy (LSM 700, Zeiss).

Image processing

Imaging data were exported to MATLAB (MathWorks, Natick, MA, USA) for post-processing. For US/PA data sets, images were manually segmented using anatomic landmarks from ultrasound to remove PA signals outside of the spinal cord. Image thresholds further eliminated noise or background signals from endogenous absorbers within the spinal cord to better visualize high PA signals from PBNC-labeled MSCs. Cross-sectional US/PA images were integrated over 1 mm in the elevational dimension to match the 1-mm slice thickness of MR images. For MR data sets, the image display was also adjusted to better visualize PBNC-labeled MSCs. After MATLAB processing, 3-D data sets were analyzed using Amira (Thermo Fisher) to display 3-D volumetric images.

For quantitative analysis of PA and MR images, the cross-sectional area of the stem cell bolus was calculated by manually segmenting the region of the spinal cord corresponding to the injection. Processing was repeated five times for each injection, and the average and standard deviation were calculated (Kubelick et al. 2020b). Cross-sectional area was calculated instead of volume because of the large 1-mm slice thickness required for MRI compared with roughly 100 μm for US/PA imaging. Given the 5-μL stem cell injection, the low spatial sampling frequency of MRI will lead to inaccurate volume calculations.

RESULTS AND DISCUSSION

US/PA and MR images were acquired up to 14 d post-injection (Fig. 1). The region corresponding to the laminectomy was clearly identified in both US and MR images based on the lack of overlying bone (Fig. 1a, 1e). Injections of PBNC-labeled MSCs were detected at days 3, 7 and 14 post-surgery with US/PA imaging at 800-nm wavelength (Fig. 1b–d) and with MRI in T2-weighted images (Fig. 1f–h). MRI served as the ground truth based on its well-established clinical use and prior results revealing detection of PBNC-labeled MSCs 24 h post-surgery (Kubelick and Emelianov 2020b). Overall, both modalities detected a similar bolus of PBNC-labeled MSCs at all time points, indicating the feasibility of using multimodal detection up to 2 wk post-operatively.

Fig. 1.

Fig. 1.

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Cross-sectional views of Prussian blue nanocube (PBNC)-labeled stem cells in the spinal cord in vivo. PBNC-Labeled stem cells were injected directly into the spinal cord of immunodeficient rats. Combined ultrasound (US, gray scale) and photoacoustic (PA, color scale) data sets were acquired with a 20-MHz ultrasound transducer and 800-nm optical wavelength. US/PA images (a–d) were post-processed to suppress background signals outside of the spinal cord US/PA (a) and MR (e) images of regions containing no injection to show anatomy. US/PA (b–d) and MR images (f–h) of the same injection site of PBNC-labeled stem cells at days 3, 7 and 14, respectively. T2-weighted MR images (f–h) serve as the ground truth. Scale Bar = 2 mm.

Both US/PA and MR images identified changes in tissue morphology over time. MRI and US at day 14 (Fig. 1d, 1h) revealed a decrease in distance between the skin and spinal cord, which likely resulted from reduced post-surgical swelling. The shorter light path caused decreased scattering of photons and increased optical fluence at the spinal cord. As a result, more PA signal corresponding to PBNC-labeled MSCs was observed at day 14 (Fig. 1d). Although our previous in vitro studies indicated a limit of detection of ∼100 cells/μL for PA versus ∼1000 cells/μL for MRI (Kubelick and Emelianov 2020a), greater imaging depth in vivo can affect the performance of PA imaging (Fig. 1d). However, PA detection of PBNC-labeled MSCs may be improved by utilizing several approaches, including background-free SAPhIRe imaging (Demissie et al. 2020) or multi-wavelength imaging and spectroscopic analysis (Kubelick et al. 2019).

In clinical studies, patients will often receive 5–10 injections (Glass et al. 2012; Feldman et al. 2014). Therefore, detecting multiple targets is an important step toward translation of the imaging approach. To assess 3-D visualization capabilities, US/PA and MRI data sets were acquired in a rat that received two injections. Volumetric images were compared at day 14 (Fig. 2a–c). Both injections of PBNC-labeled MSCs were visible with US/PA and MRI (Fig. 2b, 2c). Photographs of the excised spinal cord (Fig. 2d, 2e) also revealed two injections at similar locations observed in US/PA/MR images. Volumetric images provide better anatomic context to assess stem cell location and migration. For quantitative analysis, Figure 3a illustrates the average cross-sectional area in the middle of injection 1 calculated at days 3, 7 and 14 from US/PA and MR images. The area was approximately 0.90 mm2 at all time points for both modalities (Fig. 3a). Next, the average cross-sectional area of each injection was calculated for all five rats at day 14 (Fig. 3b). One rat received two injections. Thus, there were six injections in total. PA/MRI results revealed good agreement for each injection at day 14. Variability was observed between different injections, and cross-sectional areas ranged from 0.37 to 1.12 mm2. The variation between different injections validates the need for image guidance as consistent stem cell delivery is a common challenge among regenerative therapies (Donnelly et al. 2012).

Fig. 2.

Fig. 2.

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Three-dimensional images of multiple injections of Prussian blue nanocube (PBNC)-labeled stem cells in the spinal cord in vivo. All images were acquired at day 14 post-injection. Combined ultrasound (US, gray scale) and photoacoustic (PA, color scale) data sets were acquired with a 20-MHz ultrasound transducer and 800-nm optical wavelength. Three-dimensional US (a), US/PA (b) and MR (c) images highlight the location and spread of the stem cell bolus. Photograph of the excised spinal column and surrounding muscle (d) at day 14. Zoomed-in photograph of red box (e). R = rostral; C = caudal.

Fig. 3.

Fig. 3.

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Quantitative analysis of Prussian blue nanocube (PBNC)-labeled mesenchymal stem cell (MSC) injections. (a) Average cross-sectional area of a PBNC-labeled MSC injection at days 3, 7 and 14. (b) Average cross-sectional area of PBNC-labeled MSC injections at day 14 from each rat (n = 5). One rat received two injections. Day 14 in (a) corresponds to injection 4 in (b). Error bars represent ±one standard deviation. Solid bars = photoacoustic imaging; striped bars = magnetic resonance imaging.

Histology, performed at day 14 post-injection, provided secondary confirmation of PA and MRI results. Brightfield photomicrographs indicated the presence of blue pigment from the PBNCs that appeared to be within endocellular vesicles based on particle aggregation (Fig. 4a). PBNC-labeled MSCs were also tagged with a fluorescent dye, and green fluorescence from confocal microscopy also verified the presence of PBNC-labeled MSCs (Fig. 4b). In combination, brightfield and confocal microscopy ensured that PBNC-labeled MSCs remained in the spinal cord by day 14. Although brightfield and fluorescent photomicrographs were from consecutive tissue sections, direct co-localization of PBNCs and stem cells was not possible because the dark blue pigment from the PBNCs blocked fluorescence signals. However, results from ex vivo histologic analysis of tissue sections should only serve as secondary confirmation of in vivo results. Differences in sensitivity, imaging depth and tissue preparation required for optical microscopy can set an unfair ground truth for development of non-invasive in vivo cell tracking techniques (Kubelick et al. 2019).

Fig. 4.

Fig. 4.

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Histology of Prussian blue nanocube (PBNC)-labeled stem cells in the spinal cord at 14 d post-injection. (a) Brightfield photomicrograph of a spinal cord tissue section stained with eosin (pink) and PBNC-labeled stem cells (blue). Before injection, PBNC-labeled stem cells were also tagged with a fluorescent dye, CellTracker Green, to further confirm the presence of stem cells; that is, the stem cells were double-labeled. (b) Confocal microscopy of a spinal cord tissue section stained with 4′,6-diamidino-2-phenylindole (blue) and double-labeled stem cells (green). Bar = 40 μm.

The best validation of longitudinal US/PA detection of PBNC-labeled MSCs in the spine is comparison with MRI. US/PA and MRI have the ability to image intact tissue and use the same contrast agent to detect stem cells—the PBNCs. Perfect correspondence between US/PA and MRI should still not be expected because of inherent differences in contrast mechanism, sensitivity, image post-processing and image analysis. However, qualitative and quantitative results of the present study revealed strong agreement between US/PA and MRI for longitudinal detection of PBNC-labeled MSCs in rats in vivo.

US/PA imaging has several benefits for postoperative monitoring. For quantification, fast acquisition and higher spatial sampling frequency, US/PA imaging is more appropriate for sensitive detection of small stem cell injection volumes. In the present study, MRI acquisition took ∼5 min versus ∼1 min for single-wavelength US/PA imaging. Implementation of multi-wavelength US/PA imaging to further improve stem cell detection will increase acquisition time. However, strategies used in cardiac ultrasound can be employed to drastically expedite imaging (Cikes et al. 2014). In addition, use of a 3-D matrix array transducer instead of a linear array transducer allows acquisition of thousands of volumes per second (Provost et al. 2014). Overall, fast acquisition of US/PA imaging improves the feasibility of using longitudinal stem cell tracking in clinic by minimizing the time required for imaging procedures. Most importantly, real-time volumetric US/PA imaging can complement MRI by allowing functional monitoring of cells and tissues (Kubelick and Emelianov 2020b). Functional PA imaging can be achieved through development of PA nanosensors designed to have signal enhancement (Dragulescu-Andrasi et al. 2013), signal reduction or spectral shifts upon changes in differentiation, proliferation and stem cell viability. Distinguishing live versus dead cells is a common issue in development of stem cell tracking techniques (Ricles et al. 2014; Dhada et al. 2019). After injection, some PBNC-labeled MSCs may be dead by day 14 even though PA/MRI signal remains. Thus, creating nanoconstructs to detect stem cell viability is particularly important to address this shortcoming.

Toward clinical translation of the trimodal imaging approach, light penetration depth will be a challenge for longitudinal US/PA imaging of the spine. The required imaging depth will vary by region of the spine and patient size. At least 3 cm of penetration depth is required (Busscher et al. 2010). Skin pigmentation will also attenuate light because of melanin absorption. Lack of overlying bone improves the clinical feasibility of the approach. In addition, use of longer wavelengths, development of optimized light delivery systems and advanced imaging hardware, or implementation of sophisticated detection mechanisms, such as leveraging magnetomotive contrast, will be required to achieve necessary imaging depths (Mehrmohammadi et al. 2011; Qu et al. 2014; Kubelick and Emelianov 2020a). Until further development of advanced hardware, the US/PA/MRI approach is a valuable tool in research settings to inform design of stem cell therapies. Current proof-of-concept results in small animals are an important step to motivate future research toward a turnkey imaging approach for clinical use.

CONCLUSIONS

US/PA and MR images detected PBNC-labeled MSCs in vivo in immunodeficient rats up to 14 d after surgery. Images from both modalities revealed qualitative and quantitative agreement, and histology confirmed the presence of PBNC-labeled MSCs. Results indicated the feasibility of using the approach for in vivo post-operative detection. The US/PA/MRI approach to monitoring PBNC-labeled MSCs in the spinal cord may become a powerful imaging tool for guidance in intra- and post-operative environments. US/PA imaging may benefit MRI by providing complementary functional information using nanosensors or spectroscopic analysis in the future.

Acknowledgments

The authors thank Dr. Johannes Leisen of the Georgia Institute of Technology for his advice on the MRI studies. This work was supported in part by Grants NS112885 and the NS117613 from the National Institutes of Health.

Footnotes

Conflict of interest disclosure—The authors have no conflicts of interest.

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