*2.6. Bioluminescence*

Bioluminescence is a natural light-emitting process, produced by various organisms, which can provide a measure of localization and viability of cells and tissues [104–106]. Derived from bioluminescent species, the light-emitting oxidation reaction of luciferase (enzyme) catalysis of luciferin (substrate) can be introduced to cells. Once a substrate is introduced, transfection of luciferase-coding genes into cells primes them for light production. The light intensity can then be detected and quantitatively evaluated—known as bioluminescence imaging (BLI) [104,107]. Resolutions of BLI can accurately trace down to the molecular or cellular scale across entire organisms during in vivo tracking [104,105]. Unlike potentially harmful contrast agents, this illumination process is non-invasive, biologically compatible, and can be longitudinally monitored across cell lineages. It also avoids the high cost, low throughput, and low sensitivity of standard instrumentation, such as MRI or CT. However, current challenges include deep tissue visualization, spatial resolution, homogeneous substrate distribution, and accurate interpretation of detected signals [104,108].

Cell tracking via BLI has been used for stem cells, immune cells, and bacteria for varied tissue types [104]. For instance, using human mesoangioblasts, seeded onto decellularized esophageal scaffolds, Crowley et al. quantified cell viability, proliferation, and migration after implantation in murine models over the course of 7 days [109]. Iwano et al. visualized tumorigenic cells in deep mouse lung vasculatures and also demonstrated successful tracking of hippocampal neuronal activity, while previous luciferins were too large to penetrate [110]. Notably, these longitudinal studies have been carried out for up to 16 months in marmosets [111]. To study the immune response, Conradi et al. monitored a fibrin scaffold seeded with neonatal rat heart cells when implanted in allogeneic, syngeneic, and immunodeficient rat recipients [108]. Allogenic grafts only survived for 14 ± 1 days, while the syngeneic and immunodeficient recipients lasted over 100 days, indicating the importance of autologous cell sourcing. Ex-vivo validation and improvement of BLI is continually being performed to advance in vivo techniques as well, such as stem cell seeding on intervertebral discs in culture [112]. Therefore, BLI is known as a longitudinal, non-invasive method of tracking implanted tissues and their progeny in vivo.

To improve the tissue penetration in vivo, synthetic enzyme and substrate analogues have emerged that emit longer, near-infrared (NIR) wavelengths (λ = 650–900 nm) that are unhindered by hemoglobin and melanin absorption ranges (λ ≤ 600 nm) [110]. Used luciferases originate from fireflies, sea pansies, and photobacteria, but have relatively similar maximum emission spectrums (λ ≤ 600 nm) [104]. Luciferin analogues have also been explored. For instance, AkaLumine-HCl has achieved NIR wavelengths (λ = 677 nm) and demonstrated improved spatial sensitivity in deep lung metastases down to the single cell level, as compared to luciferin-D or CycLuc1 [110]. A mutagenic

derived luciferase, specific to AkaLumine-HCl, called Akaluc, was also engineered to boost catalysis efficiency by sevenfold. AkaLumine-HCl is permeable through the blood-brain barrier and evenly distributes at low concentrations [111]. Overall, as techniques evolve, the BLI utility in localizing tissue-engineered constructs longitudinally will continue to distinguish this method from other in vivo imaging modalities.

In a recent approach, functionalized bioinks were developed by incorporating luminescent optical sensor nanoparticles into the hydrogel ink solution [113]. Excitation of these nanoparticles with blue light results in the emission of red luminescent light by the particles which is in proportion to the local oxygen concentration. Higher oxygen contents will generate less red luminescence. Therefore, this innovative, noninvasive approach enables imaging and analysis of the heatmap of red luminescence and oxygen concentration within bioprinted tissue constructs (Figure 8) [113]. In another study, the proliferation of bioprinted mesenchymal stromal cells, associated with collagen and nano-hydroxyapatite, was assessed by quantification of the luciferase signal of luciferase positive cells in a mouse model for up to 42 days [114].

**Figure 8.** A novel 3D bioprinting approach with hydrogel bioinks functionalized with luminescent nanoparticles. (**a**) Cells and/or nanoparticles, containing the O2-sensitive luminescent indicator PtTFPP compound and an inert fluorescent coumarin dye, were incorporated into an alginate-based bioink for bioprinting. (**b**) Experimental setup used to image O2 distribution in bioprinted hydrogel constructs.
