Hesam Parsa

Real-Time Microfluidic System for Studying Mammalian Cells in 3D Microenvironments

We describe a microfluidic system that can control, in real time, the microenvironments of mammalian cells in naturally derived 3D extracellular matrix (ECM). This chip combines pneumatically actuated valves with an individually addressable array of 3D cell-laden ECM; actuation of valves determines the pathways for delivering reagents through the chip and for exchanging diffusible factors between cell chambers. To promote rapid perfusion of reagents through 3D gels (with complete exchange of reagents within the gel in seconds), we created conduits above the gels for fluid flow, and microposts to stabilize the gels under high perfusion rates. As a biological demonstration, we studied spatially segregated mouse embryonic stem cells and mouse embryonic fibroblasts embedded in 3D Matrigel over days of culture. Overall, this system may be useful for high-throughput screening, single-cell analysis and studies of cell-cell communication, where rapid control of 3D cellular microenvironments is desired.

Assembly of complex cell microenvironments using geometrically docked hydrogel shapes

Cellular communities in living tissues act in concert to establish intricate microenvironments, with complexity difficult to recapitulate in vitro. We report a method for docking numerous cellularized hydrogel shapes (100–1,000 µm in size) into hydrogel templates to construct 3D cellular microenvironments. Each shape can be uniquely designed to contain customizable concentrations of cells and molecular species, and can be placed into any spatial configuration, providing extensive compositional and geometric tunability of shape-coded patterns using a highly biocompatible hydrogel material. Using precisely arranged hydrogel shapes, we investigated migratory patterns of human mesenchymal stem cells and endothelial cells. We then developed a finite element gradient model predicting chemotactic directions of cell migration in micropatterned cocultures that were validated by tracking ∼2,500 individual cell trajectories. This simple yet robust hydrogel platform provides a comprehensive approach to the assembly of 3D cell environments.

Uncovering the behaviors of individual cells within a multicellular microvascular community.

Although individual cells vary in behavior during the formation of tissues, the nature of such variations are largely uncharacterized. Here, we tracked the morphologies and motilities of ~300 human endothelial cells from an initial dispersed state to the formation of capillary-like structures, distilling the dynamics of tissue morphogenesis into an array of ~36,000 numerical phenotypes. Quantitative analysis of population averages revealed two previously unidentified phases in which the cells spread before forming connections with neighboring cells and where the microvascular plexus stabilized before spatially reorganizing. Analysis at the single-cell level showed that in contrast to the population-averaged behavior, most cells followed distinct temporal patterns that were not reflected in the bulk average. Interestingly, some of these behavioral patterns correlated to the cells’ final structural role within the plexus. Knowledge of how individual cells or groups of cells behave enhances our understanding of how native tissues self-organize and could ultimately enable more precise approaches for engineering tissues and synthesizing multicellular communities.

Bioengineering methods for myocardial regeneration

The challenging task of heart regeneration is being pursued in three related directions: derivation of cardiomyocytes from human stem cells, in vitro engineering and maturation of cardiac tissues, and development of methods for controllable cell delivery into the heart. In this review, we focus on tissue engineering methods that recapitulate biophysical signaling found during normal heart development and maturation. We discuss the use of scaffold-bioreactor systems for engineering functional human cardiac tissues, and the methods for delivering stem cells, cardiomyocytes and engineered tissues into the heart.

Microfluidics for Engineering 3D Tissues and Cellular Microenvironments

Cellular microenvironments in native tissues are three dimensional (3D), inhomogeneous, anisotropic, and dynamic in terms of their composition of cells, extracellular matrix components, soluble factors, and physical forces (e.g. fluid flow and mechanical stress). Therefore, methods to recapitulate and control various components of cellular microenvironments in vitro and in vivo are highly useful for both studying and engineering biological systems. Microfluidics-based technologies enable precision spatiotemporal control over mass transport and are thus well suited to control the assembly and dynamic culture of engineered cellular microenvironments. In this chapter, we highlight several different ways that microfluidics can be utilized in engineering 3D tissues and cellular microenvironments, including methods to microfabricate 3D tissue scaffolds using microfluidics, methods to assemble and dynamically culture 3D microenvironments within PDMS-based microfluidic devices, and methods to incorporate microfluidic channels directly within engineered 3D tissue scaffolds, for both perfusion of tissue constructs with media and for assembly of multiphase 3D tissues.

Analyte capture in microfluidic heterogeneous immunoassays

Despite the prevalence of microfluidic-based heterogeneous immunoassays, there is incomplete understanding of analyte capture parameters. This study presents computational results and corresponding experimental binding assays of analyte capture. Our results identify: 1) a “reagent-limited” regime, under constraints of finite sample volume and assay time; 2) a critical flow rate; 3) an increase in signal by using a short concentrated plug rather than a long dilute plug; 4) a requirement to eventually reach a reaction-limited operating regime to maximize the capture of analytes [1].