Optimization of culture conditions for human bone marrow-derived mesenchymal stromal cell expansion in macrocarrier-based Tide Motion system.

BACKGROUND: With high cell doses required for mesenchymal stromal cell (MSC) clinical trials, there is a need to upgrade technologies that facilitate efficient scale up of MSCs for cell therapy. Conventional expansion with 2D culture vessels becomes the bottleneck when large cell dosages are required. Tide Motion bioreactors offer a robust, scalable platform using BioNOC II macrocarriers developed for the production of adherent cells. METHODS: We evaluated the growth and expansion of bone marrow-derived MSCs (BM-MSCs) on the macrocarrier-based culture system by optimizing key parameters such as cell seeding densities, culturing conditions, and harvesting procedures to achieve optimal cell growth. BM-MSCs expanded in conventional 2D adherent cultures were seeded into BioNOC II macrocarriers and grown in serum-containing or serum-free medium. RESULTS: BM-MSCs attained a maximum cell density of 0.49 ± 0.07 × 106 cells/carrier after 12 days of culture in BioNOC II macrocarriers with cell viability > 86% while retaining MSC specific characteristics such as surface marker expression, tri-lineage differentiation potential, immunosuppressive properties, and potency. CONCLUSION: These results reveal the feasibility of BM-MSC expansion in the scalable macrocarrier-based Tide Motion system both under serum and serum-free conditions and represent an important step for the large-scale production system of BM-MSC based cellular therapies.

MnSOD mediates shear stress-promoted tumor cell migration and adhesion.

Circulation of cancer cells in the bloodstream is a vital step for distant metastasis, during which cancer cells are exposed to hemodynamic shear stress (SS). The actions of SS on tumor cells are complicated and not fully understood. We previously reported that fluidic SS was able to promote migration of breast cancer cells by elevating the cellular ROS level. In this study, we further investigated the mechanisms regulating SS-promoted cell migration and identified the role of MnSOD in the related pathway. We found that SS could enhance tumor cell adhesion to extracellular matrix and endothelial monolayer, and MnSOD also regulated this process. Briefly, SS stimulates the generation of mitochondrial superoxide in tumor cells. MnSOD then converts superoxide into hydrogen peroxide, which activates ERK1/2 to promote tumor cell migration and activates FAK to promote tumor cell adhesion. Combining our previous and present studies, we present experimental evidence on the pro-metastatic effects of hemodynamic SS and reveal the underlying mechanism. Our findings provide new insights into the nature of cancer metastasis and the understanding of tumor cell responses to external stresses and have valuable implications for cancer therapy development.

Bioengineered three-dimensional co-culture of cancer cells and endothelial cells: A model system for dual analysis of tumor growth and angiogenesis.

Angiogenesis marks the transformation of a benign local tumor into a life-threatening disease. Many in vitro assays are available on two-dimensional (2D) platforms, however, limited research has been conducted to investigate the behavior of tumors and endothelial cells (ECs) grown on three-dimensional (3D) platforms. This study provides a 3D co-culture spheroid of tumor cells with ECs to study the interplay between ECs and tumor cells. In a 3D co-culture with HepG2 hepatocellular carcinoma (HCC) cells, ECs differentiate to form tubule networks when in co-culture. Addition of angiogenic factors or angiogenesis inhibitors to the model system enhanced or inhibited endothelial differentiation in the 3D model, enabling investigations of the cellular signaling pathways utilized in HCC development. The 3D model demonstrated similar protein expression levels as a HCC xenograft, as well as exhibited upregulation of essential signaling proteins such as Akt/mTor in the 3D model, which is not reflected in the 2D model. The effects of several anti-angiogenic agents, such as sorafenib, sunitinib, and axitinib were analyzed in the 3D co-culture model by utilizing fluorescent proteins and a fluorescence resonance energy transfer (FRET)-based caspase-3 sensor in the ECs, which can detect apoptosis in real time. The apoptotic capability of a drug to inhibit angiogenesis in the 3D model can be easily distinguished via the FRET sensor, and dual screening of anti-angiogenesis and anti-tumor drugs can be achieved in a single step via the 3D co-culture model. In summary, a 3D co-culture model is constructed, where a HCC tumor microenvironment with a hypoxic core and true gradient penetration of drugs is achieved for drug screening purposes and in vitro studies utilizing a small HCC tumor. Biotechnol. Bioeng. 2017;114: 1865-1877. © 2017 Wiley Periodicals, Inc.

Hemodynamic shear stress stimulates migration and extravasation of tumor cells by elevating cellular oxidative level.

Cancer cells are shed into the blood stream and are exposed to hemodynamic shear stress during metastasis. It has been shown that shear stress can destroy circulating tumor cells (CTCs) both in vitro and in vivo. However, it remains unclear whether shear stress can modulate the properties and functions of tumor cells in a manner that might help CTCs to exit circulation. In this study, we established a microfluidic circulatory system to apply physiological fluid shear stress on breast cancer cells and demonstrated that an arterial level of shear stress significantly enhanced tumor cell migration in transwell and wound healing assays, and enhanced extravasation in a transendothelial assay. Circulatory treatment elevated the intracellular levels of reactive oxygen species (ROS), which is an early and indispensable event for activating the extracellular signal-regulated kinases (ERK1/2). Subsequently, ERK1/2 activation promoted the migration of tumor cells and enhanced their extravasation. Finally, reducing cellular ROS production suppressed tumor cell extravasation in both a transendothelial assay and a zebrafish model. This new understanding of how fluid shear stress promotes tumor cell migration has important implications in cancer treatment and can help us to identify potential therapeutic targets for inhibiting tumor progression.

Improving electron trans-inner membrane movements in microbial electrocatalysts.

A novel nondestructive strategy of improving electron trans-inner membrane movements in bioelectrocatalysts is realized by overexpressing NADH dehydrogenase II in the inner membrane. A microbial fuel cell loaded with these improved bioelectrocatalysts shows significantly enhanced performance based on promoting the utilization of intracellular primary electron donors in bioelectrocatalysts.

Physical supports from liver cancer cells are essential for differentiation and remodeling of endothelial cells in a HepG2-HUVEC co-culture model.

Blood vessel remodeling is crucial in tumor growth. Growth factors released by tumor cells and endothelium-extracellular matrix interactions are highlighted in tumor angiogenesis, however the physical tumor-endothelium interactions are highly neglected. Here, we report that the physical supports from hepatocellular carcinoma, HepG2 cells, are essential for the differentiation and remodeling of endothelial cells. In a HepG2-HUVEC co-culture model, endothelial cells in direct contact with HepG2 cells could differentiate and form tubular structures similar to those plated on matrigel. By employing HepG2 cell sheet as a supportive layer, endothelial cells formed protrusions and sprouts above it. In separate experiments, fixed HepG2 cells could stimulate endothelial cells differentiation while the conditioned media could not, indicating that physical interactions between tumor and endothelial cells were indispensable. To further investigate the endothelium-remodeling mechanisms, the co-culture model was treated with inhibitors targeting different angiogenic signaling pathways. Inhibitors targeting focal adhesions effectively inhibited the differentiation of endothelial cells, while the growth factor receptor inhibitor displayed little effect. In conclusion, the co-culture model has provided evidences of the essential role of cancer cells in the differentiation and remodeling of endothelial cells, and is a potential platform for the discovery of new anti-angiogenic agents for liver cancer therapy.

Dual functions of gold nanorods as photothermal agent and autofluorescence enhancer to track cell death during plasmonic photothermal therapy.

Gold nanorods have the potential to localize the treatment procedure by hyperthermia and influence the fluorescence. The longitudinal plasmon peak contributes to the photothermal effect by converting light to heat. When these nanorods are PEGylated, it not only makes it biocompatible but also acts as a spacer layer during fluorescence enhancement. When the PEGylated nanorods are internalized inside the cells through endocytosis, the transverse plasmonic peak combined with the enhanced absorption and scattering properties of the nanorods can enhance the autofluorescence emission intensity from the cell. The autofluorescence from the mitochondria inside cells which reflects the respiratory status of the cell was enhanced two times by the presence of nanorods within the cell. At four minutes, the nanorods incubated cells reached the hyperthermic temperature when illuminated continuously with near infrared laser. The cell viability test and autofluorescence intensity curve showed a similar trend indicating the progress of cell death over time. This is the first report to the best of our knowledge to suggest the potential of exploiting the dual capabilities of gold nanorods as photothermal agents and autofluorescence enhancer to track cell death.