Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device.

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.

Cell behavior observation and gene expression analysis of melanoma associated with stromal fibroblasts in a three-dimensional magnetic cell culture array.

A three-dimensional (3D) multicellular tumor spheroid culture array has been fabricated using a magnetic force-based cell patterning method, analyzing the effect of stromal fibroblast on the invasive capacity of melanoma. Formation of spheroids was observed when array-like multicellular patterns of melanoma were developed using a pin-holder device made of magnetic soft iron and an external magnet, which enables the assembly of the magnetically labeled cells on the collagen gel-coated surface as array-like cell patterns. The interaction of fibroblast on the invasion of melanoma was investigated using three types of cell interaction models: (i) fibroblasts were magnetically labeled and patterned together in array with melanoma spheroids (direct-interaction model), (ii) fibroblasts coexisting in the upper collagen gel (indirect-interaction model) of melanoma spheroids, and (iii) fibroblast-sheets coexisting under melanoma spheroids (fibroblast-sheet model). The fibroblast-sheet model has largely increased the invasive capacity of melanoma, and the promotion of adhesion, migration, and invasion were also observed. In the fibroblast-sheet model, the expression of IL-8 and MMP-2 increased by 24-fold and 2-fold, respectively, in real time RT-PCR compared to the absence of fibroblasts. The results presented in this study demonstrate the importance of fibroblast interaction to invasive capacity of melanoma in the 3D in vitro bioengineered tumor microenvironment.

Magnetic force-based cell patterning for evaluation of the effect of stromal fibroblasts on invasive capacity in 3D cultures.

Biomimetic cell culture systems that recreate tumor microenvironments are necessary in understanding the progression of cancer cells in cell-to-cell interaction and in cell-to-extracellular matrix interaction. We have developed a three-dimensional spheroid array embedded in collagen for evaluation of the effect of stromal fibroblasts associated with cancer cells. When the breast epithelial cancer cell model MCF10A/myr-Akt1 was magnetically labeled and aligned in the array by an external magnetic force using a pin-holder device and a magnet, a stellate configuration was observed. Changes in MCF10A/myr-Akt1 cell behavior were only slight when normal human dermal fibroblasts (NHDF) cells coexisted in collagen (indirect-interaction array). In contrast, when NHDF were magnetically labeled and patterned together with MCF10A/myr-Akt1 (direct-interaction array), spreading and progression were observed along with NHDF. Cell image analysis indicated that the length and area were statistically significantly increased in the direct-interaction array compared to the MCF10A/myr-Akt1 alone or to the indirect-interaction array. A cell susceptibility assay was undertaken with breast cancer MDA-MB-231 associated with NHDF in the indirect-interaction array. Interestingly, although distinct suppression of cell movement and proliferation was observed with 100 µM of collagenase inhibitor, formation of invadepodia significantly increased with coexistent NHDF. Since cancer progression is influenced by its microenvironment, this magnetic cell-patterning method which clarifies direct and indirect effects of stromal cells on invasion and proliferation, is well suited for evaluation and design of more efficient approaches in cancer prevention and treatment.