The promise of cell therapy for repair and restoration of damaged tissues or organs relies on administration of large dose of cells whose healing benefits are still limited and sometimes irreproducible due to uncontrollable cell loss and death at lesion sites. Using a large amount of therapeutic cells increases the costs for cell processing and the risks of side effects. Optimal cell delivery strategies are therefore in urgent need to enhance the specificity, efficacy, and reproducibility of cell therapy leading to minimized cell dosage and side effects. Here, we addressed this unmet need by developing injectable 3D microscale cellular niches (microniches) based on biodegradable gelatin microcryogels (GMs). The microniches are constituted by in vitro priming human adipose-derived mesenchymal stem cells (hMSCs) seeded within GMs resulting in tissue-like ensembles with enriched extracellular matrices and enhanced cell-cell interactions. The primed 3D microniches facilitated cell protection from mechanical insults during injection and in vivo cell retention, survival, and ultimate therapeutic functions in treatment of critical limb ischemia (CLI) in mouse models compared with free cell-based therapy. In particular, 3D microniche-based therapy with 10 5 hMSCs realized better ischemic limb salvage than treatment with 10 6 freeinjected hMSCs, the minimum dosage with therapeutic effects for treating CLI in literature. To the best of our knowledge, this is the first convincing demonstration of injectable and primed cell delivery strategy realizing superior therapeutic efficacy for treating CLI with the lowest cell dosage in mouse models. This study offers a widely applicable cell delivery platform technology to boost the healing power of cell regenerative therapy.C ell-based regenerative therapy holds great promise for repair and restoration of damaged tissues or organs with numerous clinical trials and preclinical animal testing reported for treating complex diseases (1). Common route of cell administration for clinical cell therapy is based on either systematic administration (e.g., i.v. infusion), relying on cells homing to the lesion sites (2), or direct injection of cells into the damaged tissues (3). However, therapeutic benefits of the administered cells are still limited and sometimes irreproducible due to cell loss and cell death (4). Taking cell therapy for ischemic heart diseases as an example, only ∼5% of mesenchymal stem cells (MSCs) survived after being transplanted into an infarcted porcine heart (5). Mechanical damage during injection, high rate of cell loss and leakage to surrounding tissues, cell death due to lack of appropriate cell-cell and cell-matrix interactions in the ischemic and inflammatory lesion tissues could all contribute to poor cell retention, survival, functionality, and reproducibility of the treatment (6, 7).A rational solution to enhance the therapeutic efficacy and reproducibility of cell therapy is to administer a large dose of cells to ensure sufficient number of functional cells ...

Fluorescence-mediated tomography (FMT) is a quantitative three-dimensional imaging technique for preclinical research applications. The combination with micro-computed tomography (µCT) enables improved reconstruction and analysis. The aim of this study is to assess the potential of µCT-FMT and kinetic modeling to determine elimination and retention of typical model drugs and drug delivery systems.We selected four fluorescent probes with different but well-known biodistribution and elimination routes: Indocyanine green (ICG), hydroxyapatite-binding OsteoSense (OS), biodegradable nanogels (NG) and microbubbles (MB). µCT-FMT scans were performed in twenty BALB/c nude mice (5 per group) at 0.25, 2, 4, 8, 24, 48 and 72 h after intravenous injection. Longitudinal organ curves were determined using interactive organ segmentation software and a pharmacokinetic whole-body model was implemented and applied to compute physiological parameters describing elimination and retention.ICG demonstrated high initial hepatic uptake which decreased rapidly while intestinal accumulation appeared for around 8 hours which is in line with the known direct uptake by hepatocytes followed by hepatobiliary elimination. Complete clearance from the body was observed at 48 h. NG showed similar but slower hepatobiliary elimination because these nanoparticles require degradation before elimination can take place. OS was strongly located in the bones in addition to high signal in the bladder at 0.25 h indicating fast renal excretion. MB showed longest retention in liver and spleen and low signal in the kidneys likely caused by renal elimination or retention of fragments. Furthermore, probe retention was found in liver (MB, NG and OS), spleen (MB) and kidneys (MB and NG) at 72 h which was confirmed by ex vivo data. The kinetic model enabled robust extraction of physiological parameters from the organ curves.In summary, µCT-FMT and kinetic modeling enable differentiation of hepatobiliary and renal elimination routes and allow for the noninvasive assessment of retention sites in relevant organs including liver, kidney, bone and spleen.

Fluorescence molecular tomography (FMT) is a promising in vivo functional imaging modality in preclinical study. When solving the ill-posed FMT inverse problem, L1 regularization can preserve the details and reduce the noise in the reconstruction results effectively. Moreover, compared with the regular L1 regularization, reweighted L1 regularization is recently reported to improve the performance. In order to realize the reweighted L1 regularization for FMT, an adaptive support driven reweighted L1-regularization (ASDR-L1) algorithm is proposed in this work. This algorithm has two integral parts: an adaptive support estimate and the iteratively updated weights. In the iteratively reweighted L1-minimization sub-problem, different weights are equivalent to different regularization parameters at different locations. Thus, ASDR-L1 can be considered as a kind of spatially variant regularization methods for FMT. Physical phantom and in vivo mouse experiments were performed to validate the proposed algorithm. The results demonstrate that the proposed reweighted L1-reguarization algorithm can significantly improve the performance in terms of relative quantitation and spatial resolution.

Peripheral arterial disease (PAD) can further cause lower limb ischemia. Quantitative evaluation of the vascular perfusion in the ischemic limb contributes to diagnosis of PAD and preclinical development of new drug. In vivo time-series indocyanine green (ICG) fluorescence imaging can noninvasively monitor blood flow and has a deep tissue penetration. The perfusion rate estimated from the time-series ICG images is not enough for the evaluation of hindlimb ischemia. The information relevant to the vascular density is also important, because angiogenesis is an essential mechanism for post-ischemic recovery. In this paper, a multiparametric evaluation method is proposed for simultaneous estimation of multiple vascular perfusion parameters, including not only the perfusion rate but also the vascular perfusion density and the time-varying ICG concentration in veins. The target method is based on a mathematical model of ICG pharmacokinetics in the mouse hindlimb. The regression analysis performed on the time-series ICG images obtained from a dynamic reflectance fluorescence imaging system. The results demonstrate that the estimated multiple parameters are effective to quantitatively evaluate the vascular perfusion and distinguish hypo-perfused tissues from well-perfused tissues in the mouse hindlimb. The proposed multiparametric evaluation method could be useful for PAD diagnosis. The estimated perfusion rate and vascular perfusion density maps (left) and the time-varying ICG concentration in veins of the ankle region (right) of the normal and ischemic hindlimbs.