Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System.

BACKGROUND: The use of bone marrow-derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host disease, diabetes, ischemic cardiomyopathy and Crohn's disease, has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells must be cost-effective, safe and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures and require prolonged culture times. METHODS: We evaluated the Quantum Cell Expansion System for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results with a flask-based method currently in clinical trials. RESULTS: After only two passages, we were able to expand a mean of 6.6 × 10(8) MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts and adipocytes. The Quantum was able to generate the target cell number of 2.0 × 10(8) cells in an average of 9 fewer days and in half the number of passages required during flask-based expansion. We estimated that the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active. CONCLUSIONS: The Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared with flasks. In the Quantum, the risk of contamination is substantially reduced because of the substantial decrease in open procedures.

Synthesis and evaluation of an anti-MLC1 × anti-CD90 bispecific antibody for targeting and retaining bone-marrow-derived multipotent stromal cells in infarcted myocardium.

A key issue regarding the use of stem cells in cardiovascular regenerative medicine is their retention in target tissues. Here, we have generated and assessed a bispecific antibody heterodimer designed to improve the retention of bone-marrow-derived multipotent stromal cells (BMMSC) in cardiac tissue damaged by myocardial infarction. The heterodimer comprises an anti-human CD90 monoclonal antibody (mAb) (clone 5E10) and an anti-myosin light chain 1 (MLC1) mAb (clone MLM508) covalently cross-linked by a bis-arylhydrazone. We modified the anti-CD90 antibody with a pegylated-4-formylbenzamide moiety to a molar substitution ratio (MSR) of 2.6 and the anti-MLC1 antibody with a 6-hydrazinonicotinamide moiety to a MSR of 0.9. The covalent modifications had no significant deleterious effect on mAb epitope binding. Furthermore, the binding of anti-CD90 antibody to BMMSCs did not prevent their differentiation into adipo-, chondro-, or osteogenic lineages. Modified antibodies were combined under mild conditions (room temperature, pH 6, 1 h) in the presence of a catalyst (aniline) to allow for rapid generation of the covalent bis-arylhydrazone, which was monitored at A(354). We evaluated epitope immunoreactivity for each mAb in the construct. Flow cytometry demonstrated binding of the bispecific construct to BMMSCs that was competed by free anti-CD90 mAb, verifying that modification and cross-linking were not detrimental to the anti-CD90 complementarity-determining region. Similarly, ELISA-based assays demonstrated bispecific antibody binding to plastic-immobilized recombinant MLC1. Excess anti-MLC1 mAb competed for bispecific antibody binding. Finally, the anti-CD90 × anti-MLC1 bispecific antibody construct induced BMMSC adhesion to plastic-immobilized MLC1 that was resistant to shear stress, as measured in parallel-plate flow chamber assays. We used mAbs that bind both human antigens and the respective pig homologues. Thus, the anti-CD90 × anti-MLC1 bispecific antibody may be used in large animal studies of acute myocardial infarction and may provide a starting point for clinical studies.

Labeling Stem Cells with a New Hybrid Bismuth/Carbon Nanotube Contrast Agent for X-Ray Imaging.

The poor retention and survival of cells after transplantation to solid tissue represent a major obstacle for the effectiveness of stem cell-based therapies. The ability to track stem cells in vivo can lead to a better understanding of the biodistribution of transplanted cells, in addition to improving the analysis of stem cell therapies' outcomes. Here, we described the use of a carbon nanotube-based contrast agent (CA) for X-ray computed tomography (CT) imaging as an intracellular CA to label bone marrow-derived mesenchymal stem cells (MSCs). Porcine MSCs were labeled without observed cytotoxicity. The CA consists of a hybrid material containing ultra-short single-walled carbon nanotubes (20-80 nm in length, US-tubes) and Bi(III) oxo-salicylate clusters which contain four Bi3+ ions per cluster (Bi4C). The CA is thus abbreviated as Bi4C@US-tubes.

Surfactant-free Gd(3+)-ion-containing carbon nanotube MRI contrast agents for stem cell labeling.

There is an ever increasing interest in developing new stem cell therapies. However, imaging and tracking stem cells in vivo after transplantation remains a serious challenge. In this work, we report new, functionalized and high-performance Gd(3+)-ion-containing ultra-short carbon nanotube (US-tube) MRI contrast agent (CA) materials which are highly-water-dispersible (ca. 35 mg ml(-1)) without the need of a surfactant. The new materials have extremely high T1-weighted relaxivities of 90 (mM s)(-1) per Gd(3+) ion at 1.5 T at room temperature and have been used to safely label porcine bone-marrow-derived mesenchymal stem cells for MR imaging. The labeled cells display excellent image contrast in phantom imaging experiments, and TEM images of the labeled cells, in general, reveal small clusters of the CA material located within the cytoplasm with 10(9) Gd(3+) ions per cell.

The use of gadolinium-carbon nanostructures to magnetically enhance stem cell retention for cellular cardiomyoplasty.

In this work, the effectiveness of using Gadonanotubes (GNTs) with an external magnetic field to improve retention of transplanted adult mesenchymal stem cells (MSCs) during cellular cardiomyoplasty was evaluated. As a high-performance T1-weighted magnetic resonance imaging (MRI) cell tracking label, the GNTs are gadolinium-loaded carbon nanotube capsules that render MSCs magnetic when internalized. MSCs were internally labeled with either superparamagnetic GNTs or colloidal diamagnetic lutetium (Lu). In vitro cell rolling assays and ex vivo cardiac perfusion experiments qualitatively demonstrated increased magnetic-assisted retention of GNT-labeled MSCs. Subsequent in vivo epicardial cell injections were performed around a 1.3 T NdFeB ring magnet sutured onto the left ventricle of female juvenile pigs (n = 21). Cell dosage, magnet exposure time, and endpoints were varied to evaluate the safety and efficacy of the proposed therapy. Quantification of retained cells in collected tissues by elemental analysis (Gd or Lu) showed that the external magnet helped retain nearly three times more GNT-labeled MSCs than Lu-labeled cells. The sutured magnet was tolerated for up to 168 h; however, an inflammatory response to the magnet was noted after 48 h. These proof-of-concept studies support the feasibility and value of using GNTs as a magnetic nanoparticle facilitator to improve cell retention during cellular cardiomyoplasty.

Bismuth@US-tubes as a Potential Contrast Agent for X-ray Imaging Applications.

The encapsulation of bismuth as BiOCl/Bi2O3 within ultra-short (ca. 50 nm) single-walled carbon nanocapsules (US-tubes) has been achieved. The Bi@US-tubes have been characterized by high-resolution transmission electron microscopy (HR-TEM), energy-dispersive X-ray spectroscopy (EDS), thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. Bi@US-tubes have been used for intracellular labeling of pig bone marrow-derived mesenchymal stem cells (MSCs) to show high X-ray contrast in computed tomography (CT) cellular imaging for the first time. The relatively high contrast is achieved with low bismuth loading (2.66% by weight) within the US-tubes and without compromising cell viability. X-ray CT imaging of Bi@US-tubes-labeled MSCs showed a nearly two-fold increase in contrast enhancement when compared to unlabeled MSCs in a 100 kV CT clinical scanner. The CT signal enhancement from the Bi@US-tubes is 500 times greater than polymer-coated Bi2S3 nanoparticles and several-fold that of any clinical iodinated contrast agent (CA) at the same concentration. Our findings suggest that the Bi@US-tubes can be used as a potential new class of X-ray CT agent for stem cell labeling and possibly in vivo tracking.

Gadonanotubes as magnetic nanolabels for stem cell detection.

Stem cell-based therapies have emerged as a promising approach in regenerative medicine. In the development of such therapies, the demand for imaging technologies that permit the noninvasive monitoring of transplanted stem cells in vivo is growing. Here, we report the performance of gadolinium-containing carbon nanocapsules, or gadonanotubes (GNTs), as a new T1-weighted magnetic resonance imaging (MRI) intracellular labeling agent for pig bone marrow-derived mesenchymal stem cells (MSCs). Without the use of a transfection agent, micromolar concentrations of GNTs can deliver up to 109 Gd(3+) ions per cell without compromising cell viability, differentiation potential, proliferation pattern, and phenotype. Imaging 10 × 106 GNT-labeled MSCs demonstrates a nearly two-fold reduction in T1 relaxation time when compared to unlabeled MSCs at 1.5 T in a clinical MRI scanner, which easily permits the discrimination of GNT-labeled MSCs in a T1-weighted MR image. It is anticipated that GNTs will allow in vivo tracking of GNT-labeled MSCs, as well as other mammalian cell types, by T1-weighted imaging with greater efficacy than other current technologies now allow.