Labeling of mesenchymal stromal cells with iron oxide-poly(l-lactide) nanoparticles for magnetic resonance imaging: uptake, persistence, effects on cellular function and magnetic resonance imaging properties

Background aims. Mesenchymal stromal cells (MSC) are the focus of research in regenerative medicine aiming at the regulatory approval of these cells for specific indications. To cope with the regulatory requirements for somatic cell therapy, novel approaches that do not interfere with the natural behavior of the cells are necessary. In this context in vivo magnetic resonance imaging (MRI) of labeled MSC could be an appropriate tool. Cell labeling for MRI with a variety of different iron oxide preparations is frequently published. However, most publications lack a comprehensive assessment of the noninterference of the contrast agent with the functionality of the labeled MSC, which is a prerequisite for the validity of cell-tracking via MRI. Methods.We studied the effects of iron oxide-poly(L-lactide) nanoparticles in MSC with flow cytom-etry, transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), Prussian blue staining, CyQuant® proliferation testing, colony-forming unit-fibroblast (CFU-F) assays, flow chamber adhesion testing, immuno-logic tests and differentiation tests. Furthermore iron-labeled MSC were studied by MRI in agarose phantoms and Wistar rats. Results. It could be demonstrated that MSC show rapid uptake of nanoparticles and long-lasting intracellular persistence in the endosomal compartment. Labeling of the MSC with these particles has no influence on viability, differentiation, clonogenicity, proliferation, adhesion, phenotype and immunosuppressive properties. They show excellent MRI properties in agarose phantoms and after subcutaneous implantation in rats over several weeks. Conclusions. These particles qualify for studying MSC homing and trafficking via MRI

2017 Research & Innovation Day Program

A one day showcase of applied research, social innovation, scholarship projects and activities.https://first.fanshawec.ca/cri_cripublications/1004/thumbnail.jp

Iridium(III) hydrido N-heterocyclic carbene-phosphine complexes as catalysts in magnetization transfer reactions

[Image: see text] The hyperpolarization (HP) method signal amplification by reversible exchange (SABRE) uses para-hydrogen to sensitize substrate detection by NMR. The catalyst systems [Ir(H)(2)(IMes)(MeCN)(2)(R)]BF(4) and [Ir(H)(2)(IMes)(py)(2)(R)]BF(4) [py = pyridine; R = PCy(3) or PPh(3); IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene], which contain both an electron-donating N-heterocyclic carbene and a phosphine, are used here to catalyze SABRE. They react with acetonitrile and pyridine to produce [Ir(H)(2)(NCMe)(py)(IMes)(PPh(3))]BF(4) and [Ir(H)(2)(NCMe)(py)(IMes)(PCy(3))]BF(4), complexes that undergo ligand exchange on a time scale commensurate with observation of the SABRE effect, which is illustrated here by the observation of both pyridine and acetonitrile HP. In this study, the required symmetry breaking that underpins SABRE is provided for by the use of chemical inequivalence rather than the previously reported magnetic inequivalence. As a consequence, we show that the ligand sphere of the polarization transfer catalyst itself becomes hyperpolarized and hence that the high-sensitivity detection of a number of reaction intermediates is possible. These species include [Ir(H)(2)(NCMe)(py)(IMes)(PPh(3))]BF(4), [Ir(H)(2)(MeOH)(py)(IMes)(PPh(3))]BF(4), and [Ir(H)(2)(NCMe)(py)(2)(PPh(3))]BF(4). Studies are also described that employ the deuterium-labeled substrates CD(3)CN and C(5)D(5)N, and the labeled ligands P(C(6)D(5))(3) and IMes-d(22), to demonstrate that dramatically improved levels of HP can be achieved as a consequence of reducing proton dilution and hence polarization wastage. By a combination of these studies with experiments in which the magnetic field experienced by the sample at the point of polarization transfer is varied, confirmation of the resonance assignments is achieved. Furthermore, when [Ir(H)(2)(pyridine-h(5))(pyridine-d(5))(IMes)(PPh(3))]BF(4) is examined, its hydride ligand signals are shown to become visible through para-hydrogen-induced polarization rather than SABRE

GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSC

<div><h3>Background</h3><p>Mesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, <em>ex vivo</em> amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components.</p> <h3>Methods and Findings</h3><p>We established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform.</p> <h3>Conclusions</h3><p>The five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion.</p> </div

Schematic summary of the GMP-compliant single-step and two-step protocols used for clinical-scale isolation and expansion of MSCs from BM.

<p>Schematic summary of the GMP-compliant single-step and two-step protocols used for clinical-scale isolation and expansion of MSCs from BM.</p

Flow cytometric analysis of MSC isolated and expanded according to a GMP-grade single-step or two-step protocol options.

<p>Results of single-step MSC passage 0 (n = 15), two-step protocol 1 (TSP1), p0 (n = 8), p1 (n = 8), protocol 2 (TSP2), p0 (n = 10), p1 (n = 10), protocol 3 (TSP3), p0 (n = 6), p1 (n = 6), protocol 4 (TSP4), p0 (n = 11), p1 (n = 12) are shown as mean and SD values.</p

Flow cytometric analysis of MSC expanded according to a GMP-grade single-step or two-step protocol.

<p>Results of single-step MSC passage 0 (n = 6–13), p1 (n = 7–12) and p>1 (n = 6–12) as well as two-step MSC at p0 (n = 6) and p1 (n = 8) are shown as percent positive in Tukey’s Whisker Plots.</p

Characteristics of the different expansion protocols.

<p>PL: platelet lysate.</p

Comparison of the different protocols and their outcomes.

*<p>always rounded up to next higher integer. All values represent mean values.</p