Amy P. Wong

Partitioning microfluidic channels with hydrogel to construct tunable 3-D cellular microenvironments.

Accurate modeling of the cellular microenvironment is important for improving studies of cell biology in vitro. Here, we demonstrate a flexible method for creating a cellular microenvironment in vitro that allows (i) controlled spatial distribution (patterning) of multiple types of cells within three-dimensional (3-D) matrices of a biologically derived, thermally curable hydrogel (Matrigel) and (ii) application of gradients of soluble factors, such as cytokines, across the hydrogel. The technique uses laminar flow to divide a microchannel into multiple subchannels separated by microslabs of hydrogel. It does not require the use of UV light or photoinitiators and is compatible with cell culture in the hydrogel. This technique makes it possible to design model systems to study cellular communication mediated by the diffusion of soluble factors within 3-D matrices. Such factors can originate either from secretions of neighboring cells patterned within the microchannel, or from an external source -- e.g., a solution of growth factors injected into a subchannel. This method is particularly useful for studying cells such as those of the immune system, which are often weakly adherent and difficult to position precisely with standard systems for cell culture. We demonstrated this application by co-culturing two types of macrophage-like cells (BAC1.2F5 and LADMAC cell lines) within spatially separated regions of a slab of hydrogel. This pair of cell lines represents a simple model system for intercellular communication: the LADMAC cells produce colony-stimulating factor 1 (CSF-1), which is required by the BAC cells for survival.

Preeclampsia : 2-Methoxyestradiol Induces Cytotrophoblast Invasion and Vascular Development Specifically under Hypoxic Conditions

Inadequate invasion of the uterus by cytotrophoblasts is speculated to result in pregnancy-induced disorders such as preeclampsia. However, the molecular mechanisms that govern appropriate invasion of cytotrophoblasts are unknown. Here, we demonstrate that under low-oxygen conditions (2.5% oxygen), 2-methoxyestradiol (2-ME), which is a metabolite of estradiol and is generated by catechol-o-methyltransferase (COMT), induces invasion of cytotrophoblasts into a naturally-derived, extracellular matrix. Neither low-oxygen conditions nor 2-ME alone induces the invasion of cytotrophoblasts in this system; however, low-oxygen conditions combined with 2-ME result in the appropriate invasion of cytotrophoblasts into the extracellular matrix. Cytotrophoblast invasion under these conditions is also associated with a decrease in the expression of hypoxia-inducible factor-1alpha (HIF-1alpha), transforming growth factor-beta3 (TGF-beta3), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Pregnant COMT-deficient mice with hypoxic placentas and preeclampsia-like features demonstrate an up-regulation of HIF-1alpha, TGF-beta3, and TIMP-2 when compared with wild-type mice; normal levels are restored on administration of 2-ME, which also results in the resolution of preeclampsia-like features in these mice. Indeed, placentas from patients with preeclampsia reveal lower levels of COMT and higher levels of HIF-1alpha, TGF-beta3, and TIMP-2 when compared with those from normal pregnant women. We demonstrate that low-oxygen conditions of the placenta are a critical co-stimulator along with 2-ME for the proper invasion of cytotrophoblasts to facilitate appropriate vascular development and oxygenation during pregnancy.

Molecular topography imaging by intermembrane fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) between lipid-linked donor and acceptor molecules in two apposing lipid bilayer membranes is used to resolve topographical features at an intermembrane junction. Efficient energy transfer occurs when the membranes are apposed closely, which creates an image, or footprint, that maps the contact zone and reveals nanometer-scale topographical structures. We experimentally characterize intermembrane FRET by using a supported membrane junction consisting of a glass-supported lipid membrane, onto which a second membrane is deposited by rupture of a giant vesicle. A series of membrane junctions containing different glycolipids (phosphatidylinositol and ganglioside GM1), protein (cholera toxin), and lipid-linked polyethylene glycol are studied. The carbohydrate and protein components influence the intermembrane separation. Differential FRET efficiency is clearly distinguishable for each case. Quantitative analysis of the FRET efficiency yields measurements of intermembrane-separation distances that agree precisely with structural data on GM1 and cholera toxin. The lateral arrangement of molecular species on the membrane surface thus can be discerned by their influence on membrane spacing without the need for direct labeling of the molecule of interest. In the case of polyethylene glycol lipid-containing membrane junctions, imaging by intermembrane FRET reveals spontaneously forming patterns that are not visible in conventional fluorescence images.