Single-cell resolution of intracellular T cell Ca2+ dynamics in response to frequency-based H2O2 stimulation.

Adaptive immune cells, such as T cells, integrate information from their extracellular environment through complex signaling networks with exquisite sensitivity in order to direct decisions on proliferation, apoptosis, and cytokine production. These signaling networks are reliant on the interplay between finely tuned secondary messengers, such as Ca2+ and H2O2. Frequency response analysis, originally developed in control engineering, is a tool used for discerning complex networks. This analytical technique has been shown to be useful for understanding biological systems and facilitates identification of the dominant behaviour of the system. We probed intracellular Ca2+ dynamics in the frequency domain to investigate the complex relationship between two second messenger signaling molecules, H2O2 and Ca2+, during T cell activation with single cell resolution. Single-cell analysis provides a unique platform for interrogating and monitoring cellular processes of interest. We utilized a previously developed microfluidic device to monitor individual T cells through time while applying a dynamic input to reveal a natural frequency of the system at approximately 2.78 mHz stimulation. Although our network was much larger with more unknown connections than previous applications, we are able to derive features from our data, observe forced oscillations associated with specific amplitudes and frequencies of stimuli, and arrive at conclusions about potential transfer function fits as well as the underlying population dynamics.

Pre-TCR ligand binding impacts thymocyte development before αßTCR expression.

Adaptive cellular immunity requires accurate self- vs. nonself-discrimination to protect against infections and tumorous transformations while at the same time excluding autoimmunity. This vital capability is programmed in the thymus through selection of αßT-cell receptors (αßTCRs) recognizing peptides bound to MHC molecules (pMHC). Here, we show that the pre-TCR (preTCR), a pTα-ß heterodimer appearing before αßTCR expression, directs a previously unappreciated initial phase of repertoire selection. Contrasting with the ligand-independent model of preTCR function, we reveal through NMR and bioforce-probe analyses that the ß-subunit binds pMHC using Vß complementarity-determining regions as well as an exposed hydrophobic Vß patch characteristic of the preTCR. Force-regulated single bonds akin to those of αßTCRs but with more promiscuous ligand specificity trigger calcium flux. Thus, thymic development involves sequential ß- and then, αß-repertoire tuning, whereby preTCR interactions with self pMHC modulate early thymocyte expansion, with implications for ß-selection, immunodominant peptide recognition, and germ line-encoded MHC interaction.

A generalizable, tunable microfluidic platform for delivering fast temporally varying chemical signals to probe single-cell response dynamics.

Understanding how biological systems transduce dynamic, soluble chemical cues into physiological processes requires robust experimental tools for generating diverse temporal chemical patterns. The advent of microfluidics has seen the development of platforms for rapid fluid exchange allowing ease of changes in the cellular microenvironment and precise cell handling. Rapid exchange is important for exposing systems to temporally varying signals. However, direct coupling of macroscale fluid flow with microstructures is potentially problematic due to the high shear stresses that inevitably add confounding mechanical perturbation effects to the biological system of interest. Here, we have devised a method of translating fast and precise macroscale flows to microscale flows using a monolithically integrated perforated membrane. We integrated a high-density cell trap array for nonadherent cells that are challenging to handle under flow conditions with a soluble chemical signal generator module. The platform enables fast and repeatable switching of stimulus and buffer at low shear stresses for quantitative live, single-cell fluorescent studies. This modular design allows facile integration of any cell-handling chip design with any chemical delivery module. We demonstrate the utility of this device by characterizing heterogeneity of oscillatory response for cells exposed to alternating Ca(2+) waveforms at various periodicities. This platform enables the analysis of cell responses to chemical perturbations at a single-cell resolution that is necessary in understanding signal transduction pathways.

Enabling systems biology approaches through microfabricated systems.

With the experimental tools and knowledge that have accrued from a long history of reductionist biology, we can now start to put the pieces together and begin to understand how biological systems function as an integrated whole. Here, we describe how microfabricated tools have demonstrated promise in addressing experimental challenges in throughput, resolution, and sensitivity to support systems-based approaches to biological understanding.

A naturally derived cardiac extracellular matrix enhances cardiac progenitor cell behavior in vitro.

Myocardial infarction (MI) produces a collagen scar, altering the local microenvironment and impeding cardiac function. Cell therapy is a promising therapeutic option to replace the billions of myocytes lost following MI. Despite early successes, chronic function remains impaired and is likely a result of poor cellular retention, proliferation, and differentiation/maturation. While some efforts to deliver cells with scaffolds have attempted to address these shortcomings, they lack the natural cues required for optimal cell function. The goal of this study was to determine whether a naturally derived cardiac extracellular matrix (cECM) could enhance cardiac progenitor cell (CPC) function in vitro. CPCs were isolated via magnetic sorting of c-kit(+) cells and were grown on plates coated with either cECM or collagen I (Col). Our results show an increase in early cardiomyocyte markers on cECM compared with Col, as well as corresponding protein expression at a later time. CPCs show stronger serum-induced proliferation on cECM compared with Col, as well as increased resistance to apoptosis following serum starvation. Finally, a microfluidic adhesion assay demonstrated stronger adhesion of CPCs to cECM compared with Col. These data suggest that cECM may be optimal for CPC therapeutic delivery, as well as providing potential mechanisms to overcome the shortcomings of naked cell therapy.

Intracellular trafficking of polyamidoamine-poly(ethylene glycol) block copolymers in DNA delivery.

The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have shown immunogenicity and toxicity dangers, but synthetic vectors have lagged in transfection efficiency. Previously, we developed a modular, linear-dendritic block copolymer architecture with high gene transfection efficiency compared to commercial standards. This rationally designed system makes use of a cationic dendritic block to condense the anionic DNA and forms complexes with favorable endosomal escape properties. The linear block provides biocompatibility and protection from serum proteins, and can be functionalized with a targeting ligand. In this work, we quantitate performance of this system with respect to intracellular barriers to gene delivery using both high-throughput and traditional approaches. An image-based, high-throughput assay for endosomal escape is described and applied to the block copolymer system. Nuclear entry is demonstrated to be the most significant barrier to more efficient delivery and will be addressed in future versions of the system.