**2. Sca**ff**old Tracking Techniques**

### *2.1. Magnetic Resonance Imaging (MRI)*

Magnetic resonance imaging (MRI) is a commonly used imaging method that has broad applications in the clinical and basic research fields. Briefly, MRI is a non-invasive imaging technique that allows for pertinent information to be gathered over the entire patient body in a highly detailed manner [14]. Importantly, MRI is usually not associated with harmful radiations, allowing for repetitive scans and longitudinal studies to be performed with minimal harmful effects, which further makes this imaging method an attractive way to diagnose clinical pathologies [15]. It uses magnetic fields and radio waves often combined with contrast agents to generate highly detailed images of tissues within the body and is widely used for applications in the clinics. Recently, there has been an increasing interest for the use of MRI in various TE applications, because of the capability of the technique in producing high-resolution 3D structural scans with minimal damage to the tissue mimic [16–18]. Such 3D reconstructions can be used, via 3D bioprinting methods, to create patient and disease-specific tissue constructs for regenerative therapies (Figure 2).

**Figure 2.** Application of patient's MRI data to generate a bioprinted scaffold for organ regeneration, disease treatment, or drug delivery. MR images (**A**) of the target organ/tissue will be acquired and processed to create a 3D STL file. The model will be 3D bioprinted using various inks and scaffolds (**B**), cultured in vitro to establish the new tissue structure and vasculature (**C**), followed by implantation in vivo to repair/regenerate target tissue/organ (**D**). Reproduced with permission from Ref. [19].

Recent advances in contrast agen<sup>t</sup> design and development have made it possible to detect and track cell populations within the complex tissue-engineered constructs both in vitro and post implantation in vivo. These in vivo MRI cell-tracking processes can be performed using a variety of contrast agents, such as gadolinium, fluorine, or manganese, as well as superparamagnetic nanoparticles [20,21]. These materials are the preferred MRI contrast agents for TE applications as they are usually less cytotoxic and o ffer more reliable cellular uptake for imaging. In addition, MRI used in conjunction with di fferent functionalized nano-contrast agents and other imaging techniques has also been used to study di fferent drug release kinetics in the field of tissue engineering [22–24]. To date, MRI imaging is increasingly used in conjunction with the next-generation additive manufacturing technologies, such as bioprinting, to track cells in TE sca ffolds. MRI has been successfully used as both a diagnostic and tracking tool, which readily allows for translation of in vitro imaging processes in the basic research stage to the clinical settings [19,25].

For instance, superparamagnetic nano-sized iron oxide particles, coated with polyethylene glycol (PEG), have been used to label both rat and human T-cells in vivo, with over 90% e fficiency and without any measurable e ffects on T-cell properties [26]. Iron oxide nanoparticles were used in another study as an MRI contrast agent, to label and track collagen-based cardiac patches following implantation onto the epicardial surface of the mouse heart (Figure 3) [17,18,27,28]. T2\*-weighted MR images demonstrated the robust capability of this technique to noninvasively visualize the engineered patch device.

**Figure 3.** MR imaging of bioengineered collagen constructs used as cardiac patch to repair ischemic heart tissue. Patches were loaded with 1.5, 3.0, and 6.0 μg/mL of iron oxide nanoparticles and imaged via MRI both in vitro (**A**,**B**) and in vivo (**C**), in a mouse model. Manganese-enhanced MRI visualized the patch grafted onto healthy myocardial tissue in di fferent groups including no treatment (control) (**i**), empty patch (**ii**), nanoparticle-loaded patch (**iii**), and loaded-empty-loaded sandwich patch (**iv**). Reproduced with permission from Ref. [17].

Stem cell-derived cellular cultures used in cartilage [29], adipose [30] and heart TE [31] have also successfully used MRI in the development, characterization, and clinical translation of the sca ffolding constructs. Furthermore, MRI imaging with superparamagnetic iron oxide nanoparticles as exogenous labeling agents has been used to study di fferent stem cell dynamics for both preclinical and clinical applications [32]. As a result, with the rapid growth of various stem cell therapies, clinical MRI will be more extensively used as a robust, noninvasive bedside tool for guided administration, delivery, and tracking of transplanted cells [33]. There is also a small but significant body of work that applied MRI imaging to nanoparticle vaccine e fficacy, focusing on immune system priming and cellular activation in cancer vaccine development [34].

### *2.2. Magnetic Particle Imaging (MPI)*

Apart from MRI, superparamagnetic iron oxide nanoparticles (SPIONs) have also been used in an emergen<sup>t</sup> imaging technique such as magnetic particle imaging (MPI) through their strong magnetization (Figure 4) [35–37]. MPI, in conjunction with MRI as a paired technique, is gaining traction in clinical diagnostics due to its significant benefits over other more established techniques [36–38]. Specifically, MPI is a relatively fast imaging method, generates zero tissue background signal, and there is no attenuation of the signal correlated to organ depth, allowing for unimpeded and quantitative high-resolution imaging at any depth and location [38–40]. Recent work has advanced the technique in its potential for robust and sensitive cardiovascular imaging of healthy and diseased conditions, such as stenosis and myocardial infarcts [39,41,42] and cell tracking [43–47], which is of grea<sup>t</sup> interest in the field of TE. In particular, MPI can be useful in imaging bioprinted organ constructs, where 3D spatial arrangemen<sup>t</sup> and resolution are fundamental limitations. Further progress in MPI, as a diagnostic and basic research tool, requires advancements in imaging physics, nanoparticle synthesis, and characterization, as well as ongoing proof-of-principle imaging of small animals, TE constructs, and more broadly in human patients [48].

**Figure 4.** Hardware setup used in magnetic particle imaging (MPI) scans using SPIONs. ( **A**) The Berkeley field-free-line MPI preclinical scanner. (**B**) To form a projection image, the magnetic field (FFL) rasters across a trajectory as shown, imaging the in vivo distribution of SPIONs in a rat. Multiple such projections can reconstruct a 3D MPI image similar to CT. Reproduced with permission from Ref. [48]**.**

MPI works are based on the direct imaging of the concentration and location of the SPION tracers, using varying magnetic fields, which have high sensitivity and are capable of significant background to signal (contrast) resolution. The field has already advanced several particles (e.g., Ferumoxytol) through the FDA for chronic kidney disease induced anemia treatment [49]. Additionally, SPIONs have been shown to successfully work in patient imaging (Resovist) [50,51], and have been used to localize sentinel lymph nodes for breast cancer detection (Sienna) [52]. They have been also used for evaluation of hyperthermia-induced solid tumor removal (NanoTherm) [53]. Importantly, MPI scans are relatively safe and radiation-free, which combined with the high contrast and sensitivity imaging capabilities, o ffers critical advantages in cardiovascular imaging and cell tracking. MPI has been used both as a diagnostic tool and as an imaging platform in bioprinting-based TE, to build

tissue/organ mimics with high fidelity to their living analogs. It is capable of detecting low numbers of cells reliably [54], which opens up the technology to be used in cell-based clinical applications, such as cancer therapies and tracking stem cell-derived TE constructs [43,46,52]. Unlike traditional radiotracing contrasts, SPIONs have a half-life that is essentially unlimited, which allows researchers to track cellular localization over long time intervals (up to several months in animal models) [55].
