Recreating the perivascular niche ex vivo using a microfluidic approach.

Stem cell niches are composed of numerous microenvironmental features, including soluble and insoluble factors, cues from other cells, and the extracellular matrix (ECM), which collectively serve to maintain stem cell quiescence and promote their ability to support tissue homeostasis. A hallmark of many adult stem cell niches is their proximity to the vasculature in vivo, a feature common to neural stem cells, mesenchymal stem cells (MSCs) from bone marrow and adipose tissue, hematopoietic stem cells, and many tumor stem cells. In this study, we describe a novel 3D microfluidic device (MFD) as a model system in which to study the molecular regulation of perivascular stem cell niches. Endothelial cells (ECs) suspended within 3D fibrin gels patterned in the device adjacent to stromal cells (either fibroblasts or bone marrow-derived MSCs) executed a morphogenetic process akin to vasculogenesis, forming a primitive vascular plexus and maturing into a robust capillary network with hollow well-defined lumens. Both MSCs and fibroblasts formed pericytic associations with the ECs but promoted capillary morphogenesis with distinct kinetics. Biochemical assays within the niche revealed that the perivascular association of MSCs required interaction between their α6ß1 integrin receptor and EC-deposited laminin. These studies demonstrate the potential of this physiologically relevant ex vivo model system to study how proximity to blood vessels may influence stem cell multipotency.

Engineering microscale cellular niches for three-dimensional multicellular co-cultures.

Modeling the in vivo microenvironment typically involves placing cells in a three-dimensional (3D) extracellular matrix (ECM) in physiologically relevant context with respect to other cells. The mechanical and chemical features of 3D microenvironments play important roles in tissue engineering, tumor growth and metastasis, and in defining stem cell niches, and it is increasingly recognized that cells behave much differently when surrounded by a 3D ECM than when anchored to a 2D substrate. To create microenvironments that more closely mimic in vivo settings, here we describe a novel microfluidic device that allows multiple discrete constructs of 3D cell-laden hydrogels to be patterned in a sequence of simple steps. The microfluidic platform allows for real-time imaging of the interactions between multiple cell types exposed to both autocrine and paracrine signaling molecules, all within a 3D ECM environment. Detailed modeling determined that surface tension, hydrophobic interactions, and spatial geometry were important factors in containing the gels within distinct separate channels during the filling process. This allowed us to pattern multiple gel types side-by-side and pattern 3D gels spatially with tight dimensional control. Cells embedded in gels could be patterned by culturing MDA-MB-231 metastatic breast cancer cells and RAW 264.1 macrophage cells within distinct collagen type I and Matrigel ECM environments, respectively. Over a 7 day culture experiment, RAW cells invaded into neighboring gels containing MDA-MB-231 cells, but not into gels lacking cells. These studies demonstrate the versatility and potential of this new microfluidic platform to engineer 3D microscale architectures to investigate cell-cell and cell-matrix interactions.

Copper transporter 2 regulates intracellular copper and sensitivity to cisplatin.

Mammalian cells express two copper (Cu) influx transporters, CTR1 and CTR2. CTR1 serves as an influx transporter for both Cu and cisplatin (cDDP). In mouse embryo fibroblasts, reduction of CTR1 expression renders cells resistant to cDDP whereas reduction of CTR2 makes them hypersensitive both in vitro and in vivo. To investigate the role of CTR2 on intracellular Cu and cDDP sensitivity its expression was molecularly altered in the human epithelial 2008 cancer cell model. Intracellular exchangeable Cu(+) was measured with the fluorescent probe Coppersensor-3 (CS3). The ability of CS3 to report on changes in intracellular Cu(+) was validated by showing that Cu chelators reduced its signal, and that changes in signal accompanied alterations in expression of the major Cu influx transporter CTR1 and the two Cu efflux transporters, ATP7A and ATP7B. Constitutive knock down of CTR2 mRNA by ∼50% reduced steady-state exchangeable Cu by 22-23% and increased the sensitivity of 2008 cells by a factor of 2.6-2.9 in two separate clones. Over-expression of CTR2 increased exchangeable Cu(+) by 150% and rendered the 2008 cells 2.5-fold resistant to cDDP. The results provide evidence that CS3 can quantitatively assess changes in exchangeable Cu(+), and that CTR2 regulates both the level of exchangeable Cu(+) and sensitivity to cDDP in a model of human epithelial cancer. This study introduces CS3 and related sensors as novel tools for probing and assaying Cu-dependent sensitivity to anticancer therapeutics.