A significant barrier to the clinical translation of cellular therapies is the limited understanding of cell fate and distribution following administration in humans. While numerous biodistribution studies exist in animal models, human data remains sparse due to the invasive nature, limited resolution and short tracking windows of current imaging modalities. To bridge this gap, novel preclinical platforms such as normothermic machine perfusion (NMP) are being developed. NMP models, which replicate human physiological conditions, enable the use of non-transplantable organs as experimental platforms to assess cell engraftment and behaviour. To support such studies, labelling protocols for multiple cell lines using the tracking agent 19F-nanoparticles (19F-NP) were optimised to establish detection thresholds and imaging sensitivity across various modalities. PMA differentiated THP-1 macrophages (THP-1m) demonstrated the highest 19F-NP uptake and detectability, justifying their selection for proof-of-concept NMP cell tracking experiments.

THP-1 monocytes were differentiated into THP-1m by treating with 100 ng/mL Phorbol 12-myristate 13-acetate (PMA) for 48 hrs at 37°C. The THP-1m cells were then incubated with 19F-NPs conjugated with a Nile Red fluorophore (20 mg/mL) in serum-free RPMI 1640 medium for 18 hrs. Following labelling, 5×10⁷ THP-1m cells were cryopreserved in liquid nitrogen. Subsequently, a research human kidney was perfused ex vivo 7 hrs with a blood-based perfusate at 37°C. Throughout the perfusion, biopsies, blood and gas samples, urine output, and Contrast-Enhanced Ultrasound (CEUS) images were collected. 1 hr into perfusion, the thawed, labelled THP-1m macrophages were delivered into the arterial tap of the circuit. At the final timepoint, the organ was analysed using In Vivo Imaging System (IVIS) and Magnetic Resonance Imaging (MRI) to investigate the distribution and localisation of the THP-1ms.
 

Increased signal intensity was observed in both contrast and B mode of the CEUS images, with strongest signal localised to the renal cortex. This was observed at all timepoints up to 7 hrs during perfusion. Post-perfusion, the remaining kidney tissue was bi-sected and scanned using IVIS revealing a bright fluorescent signal throughout the cortex of the bisected kidney and not the medulla. Nile Red signal was also observed within cortical glomeruli and around medullary vessel endothelium with immunofluorescent microscopy. Co-localisation of Nile Red with the CD14 macrophage marker confirmed the signal origin to be THP-1m infused cells (figure 1). 

These findings demonstrate a proof of concept for the use of NMP as a human model system to track cell fate using multiple imaging modalities. The next phase will apply 19F-NP-labelled THP-1ms to a Multi-visceral Perfusion (MVP) model to determine whether cells can be tracked between organs over an extended perfusion period. It is hoped that this model can help streamline our understanding of cell fate avoiding the ethical and mechanistic issues associated with animal models, reducing risk to patients and unlocking the potential for cellular therapies.