Biomolecular Gradients Research Articles

Raman Spectroscopic Imaging of Cholesterol and Docosahexaenoic Acid Distribution in the Retinal Rod Outer Segment

Raman vibrational spectroscopic imaging was performed on retinal rod cells isolated from bullfrogs (Rana catesbeiana). The Raman spectra enable determination of the lipid and protein rich rod outer segment (ROS) from the nucleus and inner segment of the cell. Peak fitting analysis of spectra obtained from individual rod photoreceptor cells show characteristic vibrational modes that can be associated with cholesterol and docosahexaenoic acid containing lipids. These results provide direct observations of biomolecular gradients in the rod photoreceptor cells, which, thus far, have been based on indirect detergent extracts and histochemical analysis with indicators such as filipin. The detected biomolecules are associated with regulation of the integral membrane protein rhodopsin, and methods capable direct observation of these biomolecules offer new routes to exploring their role in the regulation of cellular processes.

A microfluidic platform for generation of sharp gradients in open-access culture

Control of the 3D microenvironment for cultured cells is essential for understanding the complex relationships that biomolecular concentration gradients have on cellular growth, regeneration, and differentiation. This paper reports a microfluidic device for delivering gradients of soluble molecules to cells in an open reservoir without exposing the cells to flow. The cells are cultured on a polyester membrane that shields them from the flow that delivers the gradient. A novel "lid" design is implemented which prevents leakage from around the membrane without requiring sealing agents or adhesives. Once layers are molded, device fabrication can be performed within minutes while at room temperature. Surface gradients were characterized with epifluorescence microscopy; image analysis verified that sharp gradients (∼33 μm wide) can be reproducibly generated. We show that heterogeneous laminar flow patterns of Orange and Green Cell Tracker (CT) applied beneath the membrane can be localized to cells cultured on the other side; concentration profile scans show the extent of CT diffusion parallel to the membrane's surface to be 10-20 μm. Our device is ideal for conventional cell culture because the cell culture surface is readily accessible to physical manipulation (e.g., micropipette access), the cell culture medium is in direct contact with the incubator atmosphere (i.e., no special protocols for ensuring proper equilibration of gas concentrations are required), and the cells are not subjected to flow-induced shear forces, which are advantageous attributes not commonly found in closed-channel microfluidic designs.

Microfluidic gradient platforms for controlling cellular behavior

Concentration gradients play an important role in controlling biological and pathological processes, such as metastasis, embryogenesis, axon guidance, and wound healing. Microfluidic devices fabricated by photo- and soft lithography techniques can manipulate the fluidic flow and diffusion profile to create biomolecular gradients in a temporal and spatial manner. Furthermore, microfluidic devices enable the control of cell-extracellular microenvironment interactions, including cell-cell, cell-matrix, and cell-soluble factor interaction. In this paper, we review the development of microfluidic-based gradient devices and highlight their biological applications.

Direct Biophotolithographic Method for Generating Substrates with Multiple Overlapping Biomolecular Patterns and Gradients

We describe an approach to generate multicomponent surface-immobilized patterns and gradients on the basis of the photochemically controlled covalent coupling of solution-phase biomolecules to benzophenone-modified substrates. Gradients were simply achieved by continuously varying the exposure to nondamaging UV light across the surface with the gradient profile controlled by biomolecule concentration and the spatial and temporal illumination of the surface. Sequential exposure of the same surface in the presence of different biomolecules resulted in overlapping patterns and gradients of proteins and carbohydrates. Finally, we preliminarily demonstrate that the resulting surfaces are suitable for generating model substrates to probe cell-substrate interactions.

Tunable Delivery Of Chemical Gradients Over Large Cell Culture Substrates Using Microfluidic Stacked Flows

Biomolecular gradients play an essential role in studying various biological phenomena such as development, cancer, inflammation, and wound healing. This paper reports a novel microfluidic device for generating tunable biomolecular gradients over large areas on cell culture surfaces. Laminar streams are stacked above the surface to generate a steady-state gradient via diffusion in the direction orthogonal to the flow and to the surface. Finite-element modelling was used to predict negligible shear forces at the range of gradients possible by tuning flow rates. The surface gradients were characterized with fluorescence microscopy; image analysis verified the presence of a one-dimensional gradient across a 2x2 mm area. Fig. 1 shows a variety of surface gradients obtained simply by changing the inlet configuration and pressure settings. Superimposed onto these images are the linescans taken across 4 different regions of the device which demonstrate lateral uniformity (excluding edge effects near walls <25% of area). Due to the binary branching of the inlets, the perfused area can be arbitrarily wide (here, 2 mm). This enables analyzing responses of large cell populations in parallel and acquiring rich statistics on single-cell variability.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Concentration gradient generator using a convective–diffusive balance

Since biomolecular concentration gradients are an important factor in various biological phenomena, many concentration gradient generators utilizing microfluidic techniques have been devised in order to establish stable in vitro biomolecular concentration gradients, each with their own strengths. However, all the fundamental issues, simplicity in apparatus, dynamic controllability, and convection-free conditions, have not been addressed simultaneously. In this paper, we describe a novel way of establishing concentration gradients using a convective-diffusive balance in a counterflow configuration. With the help of this method, stable, reproducible concentration gradient profiles can be formed in under a minute and the shape of the profiles are easily predicted with a simple mathematical formula.

Biomolecular gradients in cell culture systems

Biomolecule gradients have been shown to play roles in a wide range of biological processes including development, inflammation, wound healing, and cancer metastasis. Elucidation of these phenomena requires the ability to expose cells to biomolecule gradients that are quantifiable, controllable, and mimic those that are present in vivo. Here we review the major biological phenomena in which biomolecule gradients are employed, traditional in vitro gradient-generating methods developed over the past 50 years, and new microfluidic devices for generating gradients. Microfluidic gradient generators offer greater levels of precision, quantitation, and spatiotemporal gradient control than traditional methods, and may greatly enhance our understanding of many biological phenomena. For each method, we outline the salient features, capabilities, and applications.