- Email: [email protected]
Photoacoustic Monitoring of the Viability of Mesenchymal Stem Cells Labeled with Indocyanine Green
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Contents lists available at ScienceDirect
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Original Article
Photoacoustic Monitoring of the Viability of Mesenchymal Stem Cells Labeled With Indocyanine Green J.M. Yoo b,c , C. Yun b,c , N.Q. Bui b,c , J. Oh a,b,c , S.Y. Nam a,b,c,∗ a b c
Department of Biomedical Engineering, Pukyong National University, Busan, South Korea Interdisciplinary Program of Biomedical Mechanical & Electrical Engineering, Pukyong National University, Busan, South Korea Center for Marine-Integrated Biomedical Technology (BK21 Plus), Pukyong National University, Busan, South Korea
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• ICG can be used as an MSC labeling probe for PA imaging.
• PA imaging of MSCs showed similarity between the PA intensity and the cell viability. • PA imaging with ICG labeling is an alternative to detect MSCs viability noninvasively.
a r t i c l e
i n f o
Article history: Received 1 August 2018 Received in revised form 16 October 2018 Accepted 7 November 2018 Available online xxxx Keywords: Photoacoustic imaging Mesenchymal stem cells Cell viability Indocyanine green Contrast agents
a b s t r a c t Background: Stem cell therapy has a huge potential to enhance the recovery of damaged tissues and organs. However, it has been reported that majority of implanted stem cells cannot survive after implantation. Therefore, noninvasive monitoring of stem cell viability is essential to estimate the efficacy of stem cell therapy. However, current imaging methods have disadvantages for monitoring of stem cell viability such as cost, penetration depth, and safety. To overcome the limitations, photoacoustic imaging well known for sufficient penetration depth, relatively low cost, and non-ionizing radiation can be a novel alternative assessment method of stem cell viability. Methods: In this study, indocyanine green was used as exogenous photoacoustic contrast agents to label mesenchymal stem cells. The photoacoustic signals were acquired before and after the cell death and quantified to monitor photoacoustic signal changes related to the cell viability. Results: The fluorescence intensity changes of ICG labeled MSCs corresponded to decrease of PA intensity after cell death. Furthermore, the PA imaging of MSCs showed similarity between the PA intensity and the cell viability. Conclusion: The experimental results imply the feasibility of noninvasive detection of stem cell viability during therapeutic procedures. © 2018 AGBM. Published by Elsevier Masson SAS. All rights reserved.
1. Introduction
*
Corresponding author at: Department of Biomedical Engineering, Pukyong National University, 45, Yongso-ro, Nam-gu, Busan, 48513, South Korea. E-mail address: [email protected] (S.Y. Nam). https://doi.org/10.1016/j.irbm.2018.11.001 1959-0318/© 2018 AGBM. Published by Elsevier Masson SAS. All rights reserved.
Stem cell therapy has been used as a novel alternative for various diseases, with a potential to overcome limitations of conventional surgeries and treatments. Among different types of stem
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cells, mesenchymal stem cells (MSCs) are highlighted for clinical applications due to their availability from various sources and significant therapeutic effects on tissue regeneration promoted by differentiation, neovascularization, and paracrine effects [1]. Therefore, the MSCs have been widely used to repair damaged and injured tissues caused by different conditions including myocardial infraction, neural disorders, graft versus host diseases, and diabetes [2]. During the therapeutic procedures, cell viability is essential to ensure efficacy of the stem cell treatment, but it can often deteriorate within a short period of time after implantation in ischemic and hypoxic conditions. For instance, it has been reported that around 80–90% of MSCs could not survive within 24 hours after intravascular or intramuscular implantation [3,4]. Consequently, for most current clinical trials, large numbers of MSCs (> a few million cells per mL) are delivered to the target site, but it often comes with limited clinical effects and additional treatments due to significant rate of cell death [5]. However, the stem cell therapy with unknown numbers of viable cells may cause irreversible side effects related to tumors, infection, and immunological responses [6]. To improve efficiency, efficacy, and safety of current stem cell therapies, the viability of implanted stem cells need to be monitored noninvasively, quantitatively, and cost-effectively using clinically applicable methods. Current stem cell tracking methods, such as bioluminescence imaging, magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) have shortcomings that affect the outcome of the treatment. In bioluminescence imaging, DNA encoded luminescent proteins can cause serious genetic transformation, which restricts its clinical applications [7]. Besides possible genetic problems, bioluminescence imaging is hardly available for quantitative assessment of deeply implanted cells [8]. MRI is a useful noninvasive imaging method to track stem cells in vivo combined with exogenous contrast agents such as superparamagnetic iron oxide nanoparticles. Yet, MRI could be an additional burden in cost and time because it is relatively expensive compared to the cost for therapeutic treatments and requires long scanning time, which limits real-time assessment [9]. Although PET/CT can noninvasively represent molecular information of the implanted cells along with morphological structures, continuous monitoring of stem cells is limited due to radiation concerns as well as non-negligible scanning and processing time. Photoacoustic (PA) imaging is based on acoustic wave generation by optical excitation and subsequent energy conversion. It is relatively cost-effective and available in real-time with high spatial resolution and sensitivity. Since photoacoustic signal amplitude is linearly proportional to the optical absorption coefficients for endogenous and exogenous contrast agents such as hemoglobin, lipids, water, nanoparticles, and dyes, physiological and cellular information can be provided using the PA imaging technique [10]. Compared to other stem cell imaging methods, noninvasive, quantitative, and nonionizing properties of PA imaging can be highly beneficial for preclinical and clinical therapeutic applications with MSCs [11,12]. However, MSCs do not strongly absorb the light, so exogenous contrast agents are usually required for stem cell tracking using PA imaging. For example, metallic nanoparticles, including iron oxide nanobubble, citrate-stabilized gold nanospheres and silica-coated gold nanorods, were used to label the MSCs and amplify PA signals [10,13,14]. However, current MSC labeling suffer from clearance concerns and uncontrolled localization of the nanoparticles which hinder their clinical applications. Indocyanine green (ICG) can be an effective photoacoustic imaging agent for stem cell monitoring because of relatively high optical absorption in the near-infrared region and its diffusion out of the stem cells after cellular death. In this study, therefore, photoacoustic as well as fluorescent signals from the MSCs labeled with ICG were monitored and analyzed considering stem cell viability. The MSCs
were labeled with various concentrations of ICG to assess toxic effects on cell viability. The changes in optical absorption and fluorescent properties related to ICG labeling and induced cell death were assessed using UV–Vis spectrophotometry and live cell fluorescence imaging. In addition, photoacoustic signals were obtained and quantified from the ICG labeled MSCs before and after cell death to demonstrate the feasibility of photoacoustic monitoring of the MSC viability [13,15]. 2. Material and methods 2.1. Preparation of mesenchymal stem cells MSCs derived from mouse bone marrow (GIBCO, Invitrogen) were prepared and maintained in phenol red-free Dulbecco’s Modified Eagle Medium (DMEM/F12, GIBCO, Invitrogen) supplemented with 10% (v/v) fetal bovine serum (F6178 Sigma, Sigma-Aldrich) in a humidified 5% CO2 incubator at 37 ◦ C. The stem cells within 4–7 passages were used in all experiments. 2.2. ICG labeling of MSCs ICG (Tokyo Chemical Industry) solution was prepared by dissolving powders in DMEM/F12. The culture medium was replaced with the labeling solution to label the stem cells with 0, 0.25, 0.5, 1.0, and 2.0 mg/mL of ICG and maintained in the humidified incubator at 5% CO2 for 1 hour. After the labeling procedure, the labeling solution was discarded and the cells were washed three times with PBS. 2.3. Assessment of cell viability After ICG labeling, the labeled cells were trypsinized and precipitated by centrifuge. The viability of live cells was measured by trypan blue exclusion test in triplicate. Before and after the acquisition of PA signals from the labeled cells in the cuvette, the aliquot of cell suspension was used to assess the cell viability by counting of trypan blue-excluding cells. 2.4. Characterization of optical absorbance To assess the optical absorbance changes before and after the ICG labeling, the labeled MSCs were measured in the UV–Vis spectrophotometer (Epoch, BioTek) at the wavelength range of 300–990 nm. The stem cells (7.5 × 103 cells) were seeded and counted in 96 well plates before and after the labeling procedure. Furthermore, MSCs labeled with ICG were included in a tissue mimicking gelatin phantom to acquire and analyze PA signals from the labeled cells. 10 μL of 16% gelatin mixed with each cell suspension (3.125, 6.25, 12.5 and 25 × 103 cells/mL) was placed on the tissue-mimicking phantom. 2.5. Live cell fluorescence imaging To verify separation of ICG from the MSCs after cell death, the labeled cells were maintained in low oxygen (<1% O2 ) and serum deprivation conditions [16] in an automated microscope which supports live cell imaging as well as environment controls (Lionheart FX, BioTek). For 16 hours, fluorescence microscopic images of the MSCs labeled with 1 mg/mL of ICG were acquired at every 2 hours to continuously monitor the release of ICG from the cells in the ischemic-mimicking conditions. To quantitatively compare fluorescence signal changes in the MSCs and background, the signals from 10 different regions each inside and outside the MSCs were obtained and analyzed.
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Fig. 1. Microscopic images of (a) non-labeled MSCs and (b) MSCs labeled with 1 mg/mL of ICG. (c) Optical absorbance spectra and (d) peaks of MSCs labeled with ICG at different concentrations (0, 0.25, 0.5, 1, and 2 mg/mL).
2.6. Photoacoustic imaging For PA monitoring of the cell viability changes, the ICG labeled MSCs (3×107 cells) were trypsinized, counted and further transferred and precipitated into a modified cuvette containing 1 mL of culture media. The PA signals were acquired 10 minutes after additional 2 mL of cell culture media was added in the cuvette. As briefly described in Fig. 4, an adjustable OPO (Surelite OPO Plus, Continuum) and a Q-switched pulsed Nd:YAG laser (Surelite, Continuum) were used to supply the excitation light at a wavelength of 800 nm. The output laser beam was coupled with a multi-mode optical fiber (0.39 NA, 600 μm core diameter, BFL48-600, Thorlabs) and a plano-convex lens (50 mm focal length, LA1225-B, Thorlabs) after rejecting the pumping light by using a 665 nm long-pass colored glass filter (FGL665S, Thorlabs). The output end of the optical fiber and the ultrasound transducer (10 MHz, 0.375 inch element diameter, I3-1006-G, Harisonic) were fixed near the bottom of the cuvette. The PA signals were obtained in three-dimensional spaces including the MSCs precipitated in the cuvette. The PA signals from viable and non-viable MSCs labeled with ICG were acquired and compared before and after addition of 100 μL sodium hydroxide solution (0.5 M/mL) to induce cell death. The PA signals were acquired at least 10 minutes after transfer of the cells or induction of cell death to guarantee the precipitation of the cells in the cuvette. The obtained photoacoustic signals were post-processed, quantitatively analyzed, and converted to PA amplitude images. To verify the possibility of monitoring of the cell viability using PA imaging, the quantified PA signal changes were compared with the absorbance of the MSCs labeled with ICG measured using the UV–Vis spectrophotometer. Also, it was demonstrated that optical properties of ICG were not changed by acute change of pH at given concentrations. 3. Results and discussion Once the MSCs were cultured with the ICG labeling agent for 1 hour, the labeled MSCs clearly showed a green color while the cell morphology was similar compared to the non-labeled
Fig. 2. (a) Cell viability of MSCs labeled with different concentrations of ICG and (b) DAPI staining image of MSCs labeled with 1 mg/mL ICG.
stem cells as represented in phase-contrast microscopic images in Fig. 1(a) and 1(b). The MSCs were labeled with ICG at different concentrations of 0, 0.25, 0.5, 1, and 2 mg/mL to assess optical property changes caused by the ICG labeling. After the labeling of MSCs, absorption peaks of around a wavelength of 800 nm which increased with increasing ICG concentrations from 0.25 mg/mL to 2 mg/mL were observed as represented in Fig. 1(c) and 1(d). The viability of the mesenchymal stem cells did not significantly decrease after ICG labeling, and it was over 90% even with the highest concentration of ICG (2 mg/mL) as represented in Fig. 2(a). Also, DAPI staining images of MSCs labeled with 1 mg/mL ICG in Fig. 2(b) showed high cell viability. For the following other experiments, the ICG concentration of 1 mg/mL was used to achieve both high cell viability and detection sensitivity. The separation of ICG from the MSCs following cell death was continuously monitored using the live cell fluorescence imaging system. Low oxygen and serum deprivation conditions were maintained in the cell culture chamber to mimic the ischemic microenvironment and induce cell death. Fluorescence images of live MSCs labeled with ICG were acquired at every 2 hours as shown in Fig. 3(a). The live cell fluorescence images clearly represented the separation and the subsequent diffusion of ICG from inside to outside of the cell membrane. The quantitative analysis of the fluorescence images in Fig. 3(b) indicates that the fluorescence sig-
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Fig. 3. (a) Fluorescence images of MSCs labeled with 1 mg/mL ICG at every 2 hours and (b) fluorescence intensities obtained from inside and outside of the cells.
nals from the ICG labeled MSCs were significantly decreased while those from background regions were increased over time. Fig. 4(a) shows a block diagram of the PA imaging system for laser pulse generation, PA signal acquisition, and motion control. Before obtaining PA signals, the MSCs labeled with ICG were maintained in the cell medium for about 10 minutes and precipitated in the cuvette combined with an acoustically and optically transparent film at the bottom. Tunable OPO pulsed laser system was operated at a wavelength of 800 nm where the absorption peaks of
the labeled cells were, and the laser pulse with a pulse duration of 5 ns was irradiated through the optical fiber to generate PA signals. The PA signals were detected using the single-element ultrasound transducer focused at the depth where the precipitated cells are located. Fig. 4(b) describes how the ICG is separated from the cell membrane and diffused into the medium after cell death. The PA signals from the precipitated MSCs labeled with ICG were acquired before and 10 minutes after cell death induction. The obtained PA signals were post-processed and converted to photoacoustic amplitude images to precisely visualize the signal changes in two dimensional images. The PA amplitude images in Fig. 4(c) clearly show a severe drop in overall contrast after the cell death induction, which implies that the concentration of ICG near the bottom of the cuvette was significantly decreased by the separation of ICG from the cells and subsequent diffusion to the cell medium. In Fig. 5, the changes of fluorescent signal intensities, photoacoustic signal intensities, and cell numbers before and after the stem cell death are compared with each other. Fig. 5(a) shows significant fluorescence signal decrease from the ICG labeled MSCs and the slight increase in the background after cell death induction, indicating the separation and the subsequent diffusion of ICG to outside of the stem cells. In addition, quantitatively analyzed PA signals before and after the cell death induction were represented in Fig. 5(b). As shown in the figure, photoacoustic signal intensity of the viable MSCs labeled with ICG was approximately twice higher than that of the non-viable MSCs, which corresponds to the experimental results of the fluorescence intensity in Fig. 5(a). Furthermore, the numbers of viable MSCs used for photoacoustic signal acquisition before and after the cell death induction to compare the cell viability with the fluorescence and photoacoustic signal changes. Fig. 5(c) clearly confirms that the number of viable MSCs after the cell death induction was extremely lower than that before the induction. The offset PA amplitude of non-viable MSCs in Fig. 5(b) was not negligible and it could be resulted from low optical absorption of non-viable cells and diluted ICG.
Fig. 4. Diagrams of (a) the photoacoustic imaging system and (b) ICG diffusion from MSCs after cell death PA amplitude images (c) of PA signals from before and after death of MSCs.
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Fig. 5. (a) Fluorescence intensity changes of ICG labeled MSCs in hypoxia condition. (b) Total photoacoustic intensity and (c) the cell viability of before and after cell death induction.
Photoacoustic imaging has been well known for its excellent merits of noninvasive real-time monitoring, cost-effectiveness, relatively high spatial and temporal resolution, safety, and quantification [10,17–19]. The results of this study demonstrate the feasibility of novel noninvasive tracking method of the MSC viability. To the best our knowledge, our work is the first study that reports on photoacoustic tracking of stem cell viability with a clinically applicable contrast agent. Various imaging modalities, such as MRI, PET/CT, and other optical imaging modalities, have been applied to overcome current limitations of stem cell tracking but suffer from many drawbacks. Some approaches using exogenous labeling or transfection with a reporter gene showed the possibility of visualizing the viability of labeled cells in vivo, but they are still at preclinical stages due to safety concerns associated with reporter genes [20,21]. However, PA imaging with the ICG labeling method can be a useful tool for clinical stem cell therapies and it can noninvasively, quantitatively, and cost-effectively detect the viability as well as the number of MSCs with sufficient scalable spatial resolution (several tens of microns) and penetration depth (several centimeters) [10,12,22]. Various studies have demonstrated that PA imaging can be safely applied to the human body with appropriate laser fluence (<20 mJ/cm2 ) and the acquired photoacoustic signals from endogenous and exogenous optical observers were quantitatively analyzed [23,24]. Also, PA imaging can quantify the number of stem cells based on the relationship between the PA intensity and the cell concentration [10,11]. Therefore, PA monitoring of ICG labeled stem cells can be a useful alternative to quantitatively assess rapid cell death and the number of survived cells when implanted to the injured area. In addition to clinical applications, the PA imaging technique also can be beneficial for nondestructive measurement of the behaviors of stem cells implemented in three-dimensional tissue engineered constructs. The ICG used as the exogenous PA contrast agent in this study can be uptaken by stem cells through the endocytic pathway and released due to cytoplasmic and biliary excretion. The mechanism of uptake and secretion of ICG has been demonstrated to follow a saturable carrier-mediated transport process which can be described by the Michaelis–Menten equation [10,13,25]. Moreover, ICG has been widely used as clinical fluorescent dye, especially in angiographic diagnosis, due to its high contrast, sensitivity, safety, and low cost [26,27]. Yet, ICG does not have high fluorescence quantum yield (2.5% in water, 1.2% in blood), so a large part of the absorbed photon energy can be transferred to the non-radiative relaxation of molecules which can enhance photoacoustic wave generation after irradiation of short laser pulses [28]. Hence, there have been several reports related to photoacoustics using ICG as an exogenous imaging probe [29]. During therapeutic procedures, once MSCs labeled with an appropriate concentration of ICG are transferred to the damaged human tissue, a large number of stem cells can be stressed by hypoxic or ischemic conditions and undergo apoptotic or necrotic cell death. Consequently, ICG in the non-viable MSCs can be separated from the cells and diffused to
peripheral areas. Finally, ICG can be metabolized and excreted by the hepatic and biliary system after bounded with serum proteins, mainly globulins, and transferred via the circulatory system [30, 31]. In the previous studies, photoacoustic signals were generated from metallic nanoparticles endocytosed in the stem cells, but the nanoparticles were remained in the peripheral tissue after cell death, which restricts PA monitoring of the stem cell viability. The suggested method in this study can be used to surmount limitations of previous photoacoustic tracking methods of implanted stem cells. 4. Conclusions In this study, the presented work demonstrated that ICG can be utilized as an effective MSC labeling probe for photoacoustic imaging with sufficient detection sensitivity and low toxicity. The live cell fluorescence microscopy clearly represented the separation and the subsequent diffusion of ICG from the MSC after the cell death induction. Furthermore, the experimental results verified that PA signal changes from the MSCs labeled with ICG corresponded with the cell viability decrease. Therefore, PA imaging combined with ICG labeling method can be a novel alternative to detect of stem cell viability non-invasively during clinical therapeutic procedures, which can overcome drawbacks of current stem cell tracking methods. Disclosure of interest The authors declare that they have no known competing financial or personal relationships that could be viewed as influencing the work reported in this paper. Funding This work has been supported by Ministry of Oceans and Fisheries, Korea. Author contributions All authors attest that they meet the current International Committee of Medical Journal Editors (ICMJE) criteria for Authorship. CRediT authorship contribution statement J.M. Yoo: Conceptualization, Data curation, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft. C. Yun: Methodology, Software, Visualization, Writing - original draft. N.Q. Bui: Methodology, Software, Visualization. J. Oh: Conceptualization, Investigation, Supervision, Writing - review & editing. S.Y. Nam: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Writing - review & editing.
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Acknowledgements This research was supported by a grant from Marine Biotechnology Program (20150220) funded by Ministry of Oceans and Fisheries, Korea. References [1] Tang YL, Zhao Q, Zhang YC, Cheng L, Liu M, Shi J, et al. Autologous mesenchymal stem cell transplantation induce VEGF and neovascularization in ischemic myocardium. Regulatory Pept 2004;117(1):3–10. [2] Phinney DG, Prockop DJ. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair—current views. Stem Cells 2007;25(11):2896–902. [3] Pons J, Huang Y, Arakawa-Hoyt J, Washko D, Takagawa J, Ye J, et al. VEGF improves survival of mesenchymal stem cells in infarcted hearts. Biochem Biophys Res Commun 2008;376(2):419–22. [4] Bagi Z, Kaley G. Where have all the stem cells gone? Circ Res 2009;104(3):280–1. [5] Karp JM, Teo GSL. Mesenchymal stem cell homing: the devil is in the details. Cell Stem Cell 2009;4(3):206–16. [6] Herberts CA, Kwa MS, Hermsen HP. Risk factors in the development of stem cell therapy. J Transl Med 2011;9(1):1. [7] O’Neill K, Lyons SK, Gallagher WM, Curran KM, Byrne AT. Bioluminescent imaging: a critical tool in pre-clinical oncology research. J Pathol 2010;220(3):317–27. [8] Nam SY, Ricles LM, Suggs LJ, Emelianov SY. Imaging strategies for tissue engineering applications. Tissue Eng, Part B, Rev 2014;21(1):88–102. [9] Zhang SJ, Wu JC. Comparison of imaging techniques for tracking cardiac stem cell therapy. J Nucl Med 2007;48(12):1916–9. [10] Nam SY, Ricles LM, Suggs LJ, Emelianov SY. In vivo ultrasound and photoacoustic monitoring of mesenchymal stem cells labeled with gold nanotracers. PLoS ONE 2012;7(5):e37267. [11] Nam SY, Chung E, Suggs LJ, Emelianov SY. Combined ultrasound and photoacoustic imaging to noninvasively assess burn injury and selectively monitor a regenerative tissue-engineered construct. Tissue Eng, Part C Methods 2015;21(6):557–66. [12] Wang LV, Hu S. Photoacoustic tomography: in vivo imaging from organelles to organs. Science 2012;335(6075):1458–62. [13] Jokerst JV, Thangaraj M, Kempen PJ, Sinclair R, Gambhir SS. Photoacoustic imaging of mesenchymal stem cells in living mice via silica-coated gold nanorods. ACS Nano 2012;6(7):5920–30. [14] Lemaster JE, Chen F, Kim T, Hariri A, Jokerst JV. Development of a trimodal contrast agent for acoustic and magnetic particle imaging of stem cells. ACS Appl Nano Mater 2018;1(3):1321–31.
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Contents lists available at ScienceDirect
IRBM www.elsevier.com/locate/irbm
Original Article
Photoacoustic Monitoring of the Viability of Mesenchymal Stem Cells Labeled With Indocyanine Green J.M. Yoo b,c , C. Yun b,c , N.Q. Bui b,c , J. Oh a,b,c , S.Y. Nam a,b,c,∗ a b c
Department of Biomedical Engineering, Pukyong National University, Busan, South Korea Interdisciplinary Program of Biomedical Mechanical & Electrical Engineering, Pukyong National University, Busan, South Korea Center for Marine-Integrated Biomedical Technology (BK21 Plus), Pukyong National University, Busan, South Korea
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• ICG can be used as an MSC labeling probe for PA imaging.
• PA imaging of MSCs showed similarity between the PA intensity and the cell viability. • PA imaging with ICG labeling is an alternative to detect MSCs viability noninvasively.
a r t i c l e
i n f o
Article history: Received 1 August 2018 Received in revised form 16 October 2018 Accepted 7 November 2018 Available online xxxx Keywords: Photoacoustic imaging Mesenchymal stem cells Cell viability Indocyanine green Contrast agents
a b s t r a c t Background: Stem cell therapy has a huge potential to enhance the recovery of damaged tissues and organs. However, it has been reported that majority of implanted stem cells cannot survive after implantation. Therefore, noninvasive monitoring of stem cell viability is essential to estimate the efficacy of stem cell therapy. However, current imaging methods have disadvantages for monitoring of stem cell viability such as cost, penetration depth, and safety. To overcome the limitations, photoacoustic imaging well known for sufficient penetration depth, relatively low cost, and non-ionizing radiation can be a novel alternative assessment method of stem cell viability. Methods: In this study, indocyanine green was used as exogenous photoacoustic contrast agents to label mesenchymal stem cells. The photoacoustic signals were acquired before and after the cell death and quantified to monitor photoacoustic signal changes related to the cell viability. Results: The fluorescence intensity changes of ICG labeled MSCs corresponded to decrease of PA intensity after cell death. Furthermore, the PA imaging of MSCs showed similarity between the PA intensity and the cell viability. Conclusion: The experimental results imply the feasibility of noninvasive detection of stem cell viability during therapeutic procedures. © 2018 AGBM. Published by Elsevier Masson SAS. All rights reserved.
1. Introduction
*
Corresponding author at: Department of Biomedical Engineering, Pukyong National University, 45, Yongso-ro, Nam-gu, Busan, 48513, South Korea. E-mail address: [email protected] (S.Y. Nam). https://doi.org/10.1016/j.irbm.2018.11.001 1959-0318/© 2018 AGBM. Published by Elsevier Masson SAS. All rights reserved.
Stem cell therapy has been used as a novel alternative for various diseases, with a potential to overcome limitations of conventional surgeries and treatments. Among different types of stem
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cells, mesenchymal stem cells (MSCs) are highlighted for clinical applications due to their availability from various sources and significant therapeutic effects on tissue regeneration promoted by differentiation, neovascularization, and paracrine effects [1]. Therefore, the MSCs have been widely used to repair damaged and injured tissues caused by different conditions including myocardial infraction, neural disorders, graft versus host diseases, and diabetes [2]. During the therapeutic procedures, cell viability is essential to ensure efficacy of the stem cell treatment, but it can often deteriorate within a short period of time after implantation in ischemic and hypoxic conditions. For instance, it has been reported that around 80–90% of MSCs could not survive within 24 hours after intravascular or intramuscular implantation [3,4]. Consequently, for most current clinical trials, large numbers of MSCs (> a few million cells per mL) are delivered to the target site, but it often comes with limited clinical effects and additional treatments due to significant rate of cell death [5]. However, the stem cell therapy with unknown numbers of viable cells may cause irreversible side effects related to tumors, infection, and immunological responses [6]. To improve efficiency, efficacy, and safety of current stem cell therapies, the viability of implanted stem cells need to be monitored noninvasively, quantitatively, and cost-effectively using clinically applicable methods. Current stem cell tracking methods, such as bioluminescence imaging, magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) have shortcomings that affect the outcome of the treatment. In bioluminescence imaging, DNA encoded luminescent proteins can cause serious genetic transformation, which restricts its clinical applications [7]. Besides possible genetic problems, bioluminescence imaging is hardly available for quantitative assessment of deeply implanted cells [8]. MRI is a useful noninvasive imaging method to track stem cells in vivo combined with exogenous contrast agents such as superparamagnetic iron oxide nanoparticles. Yet, MRI could be an additional burden in cost and time because it is relatively expensive compared to the cost for therapeutic treatments and requires long scanning time, which limits real-time assessment [9]. Although PET/CT can noninvasively represent molecular information of the implanted cells along with morphological structures, continuous monitoring of stem cells is limited due to radiation concerns as well as non-negligible scanning and processing time. Photoacoustic (PA) imaging is based on acoustic wave generation by optical excitation and subsequent energy conversion. It is relatively cost-effective and available in real-time with high spatial resolution and sensitivity. Since photoacoustic signal amplitude is linearly proportional to the optical absorption coefficients for endogenous and exogenous contrast agents such as hemoglobin, lipids, water, nanoparticles, and dyes, physiological and cellular information can be provided using the PA imaging technique [10]. Compared to other stem cell imaging methods, noninvasive, quantitative, and nonionizing properties of PA imaging can be highly beneficial for preclinical and clinical therapeutic applications with MSCs [11,12]. However, MSCs do not strongly absorb the light, so exogenous contrast agents are usually required for stem cell tracking using PA imaging. For example, metallic nanoparticles, including iron oxide nanobubble, citrate-stabilized gold nanospheres and silica-coated gold nanorods, were used to label the MSCs and amplify PA signals [10,13,14]. However, current MSC labeling suffer from clearance concerns and uncontrolled localization of the nanoparticles which hinder their clinical applications. Indocyanine green (ICG) can be an effective photoacoustic imaging agent for stem cell monitoring because of relatively high optical absorption in the near-infrared region and its diffusion out of the stem cells after cellular death. In this study, therefore, photoacoustic as well as fluorescent signals from the MSCs labeled with ICG were monitored and analyzed considering stem cell viability. The MSCs
were labeled with various concentrations of ICG to assess toxic effects on cell viability. The changes in optical absorption and fluorescent properties related to ICG labeling and induced cell death were assessed using UV–Vis spectrophotometry and live cell fluorescence imaging. In addition, photoacoustic signals were obtained and quantified from the ICG labeled MSCs before and after cell death to demonstrate the feasibility of photoacoustic monitoring of the MSC viability [13,15]. 2. Material and methods 2.1. Preparation of mesenchymal stem cells MSCs derived from mouse bone marrow (GIBCO, Invitrogen) were prepared and maintained in phenol red-free Dulbecco’s Modified Eagle Medium (DMEM/F12, GIBCO, Invitrogen) supplemented with 10% (v/v) fetal bovine serum (F6178 Sigma, Sigma-Aldrich) in a humidified 5% CO2 incubator at 37 ◦ C. The stem cells within 4–7 passages were used in all experiments. 2.2. ICG labeling of MSCs ICG (Tokyo Chemical Industry) solution was prepared by dissolving powders in DMEM/F12. The culture medium was replaced with the labeling solution to label the stem cells with 0, 0.25, 0.5, 1.0, and 2.0 mg/mL of ICG and maintained in the humidified incubator at 5% CO2 for 1 hour. After the labeling procedure, the labeling solution was discarded and the cells were washed three times with PBS. 2.3. Assessment of cell viability After ICG labeling, the labeled cells were trypsinized and precipitated by centrifuge. The viability of live cells was measured by trypan blue exclusion test in triplicate. Before and after the acquisition of PA signals from the labeled cells in the cuvette, the aliquot of cell suspension was used to assess the cell viability by counting of trypan blue-excluding cells. 2.4. Characterization of optical absorbance To assess the optical absorbance changes before and after the ICG labeling, the labeled MSCs were measured in the UV–Vis spectrophotometer (Epoch, BioTek) at the wavelength range of 300–990 nm. The stem cells (7.5 × 103 cells) were seeded and counted in 96 well plates before and after the labeling procedure. Furthermore, MSCs labeled with ICG were included in a tissue mimicking gelatin phantom to acquire and analyze PA signals from the labeled cells. 10 μL of 16% gelatin mixed with each cell suspension (3.125, 6.25, 12.5 and 25 × 103 cells/mL) was placed on the tissue-mimicking phantom. 2.5. Live cell fluorescence imaging To verify separation of ICG from the MSCs after cell death, the labeled cells were maintained in low oxygen (<1% O2 ) and serum deprivation conditions [16] in an automated microscope which supports live cell imaging as well as environment controls (Lionheart FX, BioTek). For 16 hours, fluorescence microscopic images of the MSCs labeled with 1 mg/mL of ICG were acquired at every 2 hours to continuously monitor the release of ICG from the cells in the ischemic-mimicking conditions. To quantitatively compare fluorescence signal changes in the MSCs and background, the signals from 10 different regions each inside and outside the MSCs were obtained and analyzed.
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Fig. 1. Microscopic images of (a) non-labeled MSCs and (b) MSCs labeled with 1 mg/mL of ICG. (c) Optical absorbance spectra and (d) peaks of MSCs labeled with ICG at different concentrations (0, 0.25, 0.5, 1, and 2 mg/mL).
2.6. Photoacoustic imaging For PA monitoring of the cell viability changes, the ICG labeled MSCs (3×107 cells) were trypsinized, counted and further transferred and precipitated into a modified cuvette containing 1 mL of culture media. The PA signals were acquired 10 minutes after additional 2 mL of cell culture media was added in the cuvette. As briefly described in Fig. 4, an adjustable OPO (Surelite OPO Plus, Continuum) and a Q-switched pulsed Nd:YAG laser (Surelite, Continuum) were used to supply the excitation light at a wavelength of 800 nm. The output laser beam was coupled with a multi-mode optical fiber (0.39 NA, 600 μm core diameter, BFL48-600, Thorlabs) and a plano-convex lens (50 mm focal length, LA1225-B, Thorlabs) after rejecting the pumping light by using a 665 nm long-pass colored glass filter (FGL665S, Thorlabs). The output end of the optical fiber and the ultrasound transducer (10 MHz, 0.375 inch element diameter, I3-1006-G, Harisonic) were fixed near the bottom of the cuvette. The PA signals were obtained in three-dimensional spaces including the MSCs precipitated in the cuvette. The PA signals from viable and non-viable MSCs labeled with ICG were acquired and compared before and after addition of 100 μL sodium hydroxide solution (0.5 M/mL) to induce cell death. The PA signals were acquired at least 10 minutes after transfer of the cells or induction of cell death to guarantee the precipitation of the cells in the cuvette. The obtained photoacoustic signals were post-processed, quantitatively analyzed, and converted to PA amplitude images. To verify the possibility of monitoring of the cell viability using PA imaging, the quantified PA signal changes were compared with the absorbance of the MSCs labeled with ICG measured using the UV–Vis spectrophotometer. Also, it was demonstrated that optical properties of ICG were not changed by acute change of pH at given concentrations. 3. Results and discussion Once the MSCs were cultured with the ICG labeling agent for 1 hour, the labeled MSCs clearly showed a green color while the cell morphology was similar compared to the non-labeled
Fig. 2. (a) Cell viability of MSCs labeled with different concentrations of ICG and (b) DAPI staining image of MSCs labeled with 1 mg/mL ICG.
stem cells as represented in phase-contrast microscopic images in Fig. 1(a) and 1(b). The MSCs were labeled with ICG at different concentrations of 0, 0.25, 0.5, 1, and 2 mg/mL to assess optical property changes caused by the ICG labeling. After the labeling of MSCs, absorption peaks of around a wavelength of 800 nm which increased with increasing ICG concentrations from 0.25 mg/mL to 2 mg/mL were observed as represented in Fig. 1(c) and 1(d). The viability of the mesenchymal stem cells did not significantly decrease after ICG labeling, and it was over 90% even with the highest concentration of ICG (2 mg/mL) as represented in Fig. 2(a). Also, DAPI staining images of MSCs labeled with 1 mg/mL ICG in Fig. 2(b) showed high cell viability. For the following other experiments, the ICG concentration of 1 mg/mL was used to achieve both high cell viability and detection sensitivity. The separation of ICG from the MSCs following cell death was continuously monitored using the live cell fluorescence imaging system. Low oxygen and serum deprivation conditions were maintained in the cell culture chamber to mimic the ischemic microenvironment and induce cell death. Fluorescence images of live MSCs labeled with ICG were acquired at every 2 hours as shown in Fig. 3(a). The live cell fluorescence images clearly represented the separation and the subsequent diffusion of ICG from inside to outside of the cell membrane. The quantitative analysis of the fluorescence images in Fig. 3(b) indicates that the fluorescence sig-
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Fig. 3. (a) Fluorescence images of MSCs labeled with 1 mg/mL ICG at every 2 hours and (b) fluorescence intensities obtained from inside and outside of the cells.
nals from the ICG labeled MSCs were significantly decreased while those from background regions were increased over time. Fig. 4(a) shows a block diagram of the PA imaging system for laser pulse generation, PA signal acquisition, and motion control. Before obtaining PA signals, the MSCs labeled with ICG were maintained in the cell medium for about 10 minutes and precipitated in the cuvette combined with an acoustically and optically transparent film at the bottom. Tunable OPO pulsed laser system was operated at a wavelength of 800 nm where the absorption peaks of
the labeled cells were, and the laser pulse with a pulse duration of 5 ns was irradiated through the optical fiber to generate PA signals. The PA signals were detected using the single-element ultrasound transducer focused at the depth where the precipitated cells are located. Fig. 4(b) describes how the ICG is separated from the cell membrane and diffused into the medium after cell death. The PA signals from the precipitated MSCs labeled with ICG were acquired before and 10 minutes after cell death induction. The obtained PA signals were post-processed and converted to photoacoustic amplitude images to precisely visualize the signal changes in two dimensional images. The PA amplitude images in Fig. 4(c) clearly show a severe drop in overall contrast after the cell death induction, which implies that the concentration of ICG near the bottom of the cuvette was significantly decreased by the separation of ICG from the cells and subsequent diffusion to the cell medium. In Fig. 5, the changes of fluorescent signal intensities, photoacoustic signal intensities, and cell numbers before and after the stem cell death are compared with each other. Fig. 5(a) shows significant fluorescence signal decrease from the ICG labeled MSCs and the slight increase in the background after cell death induction, indicating the separation and the subsequent diffusion of ICG to outside of the stem cells. In addition, quantitatively analyzed PA signals before and after the cell death induction were represented in Fig. 5(b). As shown in the figure, photoacoustic signal intensity of the viable MSCs labeled with ICG was approximately twice higher than that of the non-viable MSCs, which corresponds to the experimental results of the fluorescence intensity in Fig. 5(a). Furthermore, the numbers of viable MSCs used for photoacoustic signal acquisition before and after the cell death induction to compare the cell viability with the fluorescence and photoacoustic signal changes. Fig. 5(c) clearly confirms that the number of viable MSCs after the cell death induction was extremely lower than that before the induction. The offset PA amplitude of non-viable MSCs in Fig. 5(b) was not negligible and it could be resulted from low optical absorption of non-viable cells and diluted ICG.
Fig. 4. Diagrams of (a) the photoacoustic imaging system and (b) ICG diffusion from MSCs after cell death PA amplitude images (c) of PA signals from before and after death of MSCs.
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Fig. 5. (a) Fluorescence intensity changes of ICG labeled MSCs in hypoxia condition. (b) Total photoacoustic intensity and (c) the cell viability of before and after cell death induction.
Photoacoustic imaging has been well known for its excellent merits of noninvasive real-time monitoring, cost-effectiveness, relatively high spatial and temporal resolution, safety, and quantification [10,17–19]. The results of this study demonstrate the feasibility of novel noninvasive tracking method of the MSC viability. To the best our knowledge, our work is the first study that reports on photoacoustic tracking of stem cell viability with a clinically applicable contrast agent. Various imaging modalities, such as MRI, PET/CT, and other optical imaging modalities, have been applied to overcome current limitations of stem cell tracking but suffer from many drawbacks. Some approaches using exogenous labeling or transfection with a reporter gene showed the possibility of visualizing the viability of labeled cells in vivo, but they are still at preclinical stages due to safety concerns associated with reporter genes [20,21]. However, PA imaging with the ICG labeling method can be a useful tool for clinical stem cell therapies and it can noninvasively, quantitatively, and cost-effectively detect the viability as well as the number of MSCs with sufficient scalable spatial resolution (several tens of microns) and penetration depth (several centimeters) [10,12,22]. Various studies have demonstrated that PA imaging can be safely applied to the human body with appropriate laser fluence (<20 mJ/cm2 ) and the acquired photoacoustic signals from endogenous and exogenous optical observers were quantitatively analyzed [23,24]. Also, PA imaging can quantify the number of stem cells based on the relationship between the PA intensity and the cell concentration [10,11]. Therefore, PA monitoring of ICG labeled stem cells can be a useful alternative to quantitatively assess rapid cell death and the number of survived cells when implanted to the injured area. In addition to clinical applications, the PA imaging technique also can be beneficial for nondestructive measurement of the behaviors of stem cells implemented in three-dimensional tissue engineered constructs. The ICG used as the exogenous PA contrast agent in this study can be uptaken by stem cells through the endocytic pathway and released due to cytoplasmic and biliary excretion. The mechanism of uptake and secretion of ICG has been demonstrated to follow a saturable carrier-mediated transport process which can be described by the Michaelis–Menten equation [10,13,25]. Moreover, ICG has been widely used as clinical fluorescent dye, especially in angiographic diagnosis, due to its high contrast, sensitivity, safety, and low cost [26,27]. Yet, ICG does not have high fluorescence quantum yield (2.5% in water, 1.2% in blood), so a large part of the absorbed photon energy can be transferred to the non-radiative relaxation of molecules which can enhance photoacoustic wave generation after irradiation of short laser pulses [28]. Hence, there have been several reports related to photoacoustics using ICG as an exogenous imaging probe [29]. During therapeutic procedures, once MSCs labeled with an appropriate concentration of ICG are transferred to the damaged human tissue, a large number of stem cells can be stressed by hypoxic or ischemic conditions and undergo apoptotic or necrotic cell death. Consequently, ICG in the non-viable MSCs can be separated from the cells and diffused to
peripheral areas. Finally, ICG can be metabolized and excreted by the hepatic and biliary system after bounded with serum proteins, mainly globulins, and transferred via the circulatory system [30, 31]. In the previous studies, photoacoustic signals were generated from metallic nanoparticles endocytosed in the stem cells, but the nanoparticles were remained in the peripheral tissue after cell death, which restricts PA monitoring of the stem cell viability. The suggested method in this study can be used to surmount limitations of previous photoacoustic tracking methods of implanted stem cells. 4. Conclusions In this study, the presented work demonstrated that ICG can be utilized as an effective MSC labeling probe for photoacoustic imaging with sufficient detection sensitivity and low toxicity. The live cell fluorescence microscopy clearly represented the separation and the subsequent diffusion of ICG from the MSC after the cell death induction. Furthermore, the experimental results verified that PA signal changes from the MSCs labeled with ICG corresponded with the cell viability decrease. Therefore, PA imaging combined with ICG labeling method can be a novel alternative to detect of stem cell viability non-invasively during clinical therapeutic procedures, which can overcome drawbacks of current stem cell tracking methods. Disclosure of interest The authors declare that they have no known competing financial or personal relationships that could be viewed as influencing the work reported in this paper. Funding This work has been supported by Ministry of Oceans and Fisheries, Korea. Author contributions All authors attest that they meet the current International Committee of Medical Journal Editors (ICMJE) criteria for Authorship. CRediT authorship contribution statement J.M. Yoo: Conceptualization, Data curation, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft. C. Yun: Methodology, Software, Visualization, Writing - original draft. N.Q. Bui: Methodology, Software, Visualization. J. Oh: Conceptualization, Investigation, Supervision, Writing - review & editing. S.Y. Nam: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Writing - review & editing.
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