Anna Tourovskaia

Differentiation-on-a-chip: A microfluidic platform for long-term cell culture studies

Here we demonstrate a microfluidic perfusion system suitable for a long-term (>2 week) culture of muscle cells spanning the whole process of differentiation from myoblasts to myotubes. Cell-adhesive surface microdomains alternating with a robust cell-repellent coating mimic in vivo spatial cues for muscle cell assembly and allow for confining the fusion of myoblasts into aligned, isolated multinucleated myotubes. The microfluidic system provides accurate control of the perfusion rates and biochemical composition of the environment surrounding the cells. Comparing muscle cell-specific differentiation markers and the timing of fusion, we observed no differences in differentiation between microfluidic and traditional cultures. All differentiation assays were fully microfluidic, i.e. they were performed by sequentially changing the fluids in the micro-channels. By delivering fluorescent markers using heterogeneous laminar flows, it was possible to confine a membrane receptor labeling assay to a region smaller than a myotube. Our method can serve as an improved in vitro model for studying muscle cell differentiation and for characterizing extracellular molecules and mechanisms involved in neuromuscular differentiation.

Tissue-engineered microenvironment systems for modeling human vasculature

The high attrition rate of drug candidates late in the development process has led to an increasing demand for test assays that predict clinical outcome better than conventional 2D cell culture systems and animal models. Government agencies, the military, and the pharmaceutical industry have started initiatives for the development of novel in-vitro systems that recapitulate functional units of human tissues and organs. There is growing evidence that 3D cell arrangement, co-culture of different cell types, and physico-chemical cues lead to improved predictive power. A key element of all tissue microenvironments is the vasculature. Beyond transporting blood the microvasculature assumes important organ-specific functions. It is also involved in pathologic conditions, such as inflammation, tumor growth, metastasis, and degenerative diseases. To provide a tool for modeling this important feature of human tissue microenvironments, we developed a microfluidic chip for creating tissue-engineered microenvironment systems (TEMS) composed of tubular cell structures. Our chip design encompasses a small chamber that is filled with an extracellular matrix (ECM) surrounding one or more tubular channels. Endothelial cells (ECs) seeded into the channels adhere to the ECM walls and grow into perfusable tubular tissue structures that are fluidically connected to upstream and downstream fluid channels in the chip. Using these chips we created models of angiogenesis, the blood–brain barrier (BBB), and tumor-cell extravasation. Our angiogenesis model recapitulates true angiogenesis, in which sprouting occurs from a “parent” vessel in response to a gradient of growth factors. Our BBB model is composed of a microvessel generated from brain-specific ECs within an ECM populated with astrocytes and pericytes. Our tumor-cell extravasation model can be utilized to visualize and measure tumor-cell migration through vessel walls into the surrounding matrix. The described technology can be used to create TEMS that recapitulate structural, functional, and physico-chemical elements of vascularized human tissue microenvironments in vitro.

Long-term microfluidic cultures of myotube microarrays for high-throughput focal stimulation

We have developed a microfluidic cell culture method that allows for the formation of linear isolated myotubes organized in a parallel microarray. Attachment and spreading of cells are confined within microtracks of cell-adherent proteins separated by a protein-repellent coating. Signaling molecules or other molecules of interest can be focally delivered to the myotubes using heterogeneous microfluidic streams. We have used the method to focally deliver agrin (a molecule implicated as a postsynaptic organizer), which leads to localized acetylcholine receptor clustering. These techniques can be modified to accommodate other cell types and can be adapted to virtually any bioactive molecule such as signaling factors or drugs. This protocol features two major techniques that can be utilized simultaneously or independently to (i) micropattern cells using surface chemical modification and (ii) use a microfluidic platform for culturing and focal stimulation of cells with molecules of interest. Device design, fabrication and assembly can be completed in 3 days.

Dual-modal three-dimensional imaging of single cells with isometric high resolution using an optical projection tomography microscope

The practice of clinical cytology relies on bright-field microscopy using absorption dyes like hematoxylin and eosin in the transmission mode, while the practice of research microscopy relies on fluorescence microscopy in the epi-illumination mode. The optical projection tomography microscope is an optical microscope that can generate 3-D images of single cells with isometric high resolution both in absorption and fluorescence mode. Although the depth of field of the microscope objective is in the submicron range, it can be extended by scanning the objectives focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. Cells suspended in optical gel flow through a custom-designed microcapillary. Multiple pseudoprojection images are taken by rotating the microcapillary. After these pseudoprojection images are further aligned, computed tomography methods are applied to create 3-D reconstruction. 3-D reconstructed images of single cells are shown in both absorption and fluorescence mode. Fluorescence spatial resolution is measured at 0.35 microm in both axial and lateral dimensions. Since fluorescence and absorption images are taken in two different rotations, mechanical error may cause misalignment of 3-D images. This mechanical error is estimated to be within the resolution of the system.

Localized Acetylcholine Receptor Clustering Dynamics in Response to Microfluidic Focal Stimulation with Agrin

Agrin is a proteoglycan secreted by the motor neurons growing axon terminal upon contact with the muscle during embryonic development. It was long thought that agrins role was to trigger the clustering of acetylcholine receptors (AChRs) to nascent synapse sites. However, agrin-predating, protosynaptic AChR clusters are present well before innervation in the embryo and in myotube cultures, yet no role has been conclusively ascribed to agrin. We used a microfluidic device to focally deliver agrin to protosynaptic AChR clusters in micropatterned myotube cultures. The distribution of AChRs labeled with fluorescent bungarotoxin was imaged at various time points over >24 h. We find that a 4-h focal application of agrin (100 nM) preferentially reduces AChR loss at agrin-exposed clusters by 17% relative to the agrin-deprived clusters on the same myotube. In addition, the focal application increases the addition of AChRs preferentially at the clusters by 10% relative to the agrin-exposed, noncluster areas. Taken together, these findings suggest that a focal agrin stimulus can play a key stabilizing role in the aggregation of AChRs at the early stages of synapse formation. This methodology is generally applicable to various developmental processes and cell types, including neurons and stem cells.

Innovations in preclinical biology: ex vivo engineering of a human kidney tissue microperfusion system

Kidney disease is a public health problem that affects more than 20 million people in the US adult population, yet little is understood about the impact of kidney disease on drug disposition. Consequently there is a critical need to be able to model the human kidney and other organ systems, to improve our understanding of drug efficacy, safety, and toxicity, especially during drug development. The kidneys in general, and the proximal tubule specifically, play a central role in the elimination of xenobiotics. With recent advances in molecular investigation, considerable information has been gathered regarding the substrate profiles of the individual transporters expressed in the proximal tubule. However, we have little knowledge of how these transporters coupled with intracellular enzymes and influenced by metabolic pathways form an efficient secretory and reabsorptive mechanism in the renal tubule. Proximal tubular secretion and reabsorption of xenobiotics is critically dependent on interactions with peritubular capillaries and the interstitium. We plan to robustly model the human kidney tubule interstitium, utilizing an ex vivo three-dimensional modular microphysiological system with human kidney-derived cells. The microphysiological system should accurately reflect human physiology, be usable to predict renal handling of xenobiotics, and should assess mechanisms of kidney injury, and the biological response to injury, from endogenous and exogenous intoxicants.

Long-term micropatterned cell cultures in heterogeneous microfluidic environments

Microfluidic poly(dimethylsiloxane) (PDMS) devices were constructed and used as long-term cell culture platforms for skeletal muscle cell differentiation and for dynamic application of chemical stimuli to the cells. The devices featured two orthogonal fluidic networks: one for long-term cell perfusion at minimal rates and the other one for short-term selective cell treatment and stimulation with biologically relevant molecules. The cells were micropatterned within the microfluidic channels using surface modification techniques, cultured under continuous flow, and allowed to fuse into polynucleated myotubes (a major milestone in muscle cell differentiation). By exposing cells to heterogeneous laminar flows, it was possible to confine a membrane receptor labeling assay to a region smaller than a cell.

Combinatorial microfluidic devices for cell biology

Cell responses to signaling factors are complex and non-linear, depending on concentration, onset and duration of exposure, and interactions with other factors. Present methods for exposing cells to soluble factors are resource-intensive and/or have low throughput. To improve combinatorial testing of single cells in culture, we are developing a novel strategy to deliver combinations of reagents to large numbers of single cells simultaneously. The core device is a passive microfluidic combinatorial mixer, which generates combinations of compounds in a large number of known concentrations from a few inputs. Device prototypes are demonstrated in laminated Mylar. Cell islands are created using protein patterns defined by holes in an elastomeric stencil.

Multimodal three-dimensional imaging with isometric high resolution using optical projection tomography

The optical projection tomography microscope (OPTM) is an optical microscope that acquires focus-invariant images from multiple views of single cells. Although the depth of field of the objective is short, it can be extended by scanning the objectives focal plane. This extended depth of field image is similar to a projection in conventional X-ray CT. Samples flow through a microcapillary tube filled with optical gel. Optical distortion is minimized by matching refractive index of optical gel and tube. Multiple projection images are taken by rotating the microcapillary tube with sub-micron mechanical precision. After these pseudoprojection images are further aligned, computed tomography methods are then applied to the images to create a 3D reconstruction with isometric resolution of 0.35 microns. Three-dimensional reconstructed images of fluorescent microspheres and cells are shown.

Cell Biology on a Chip: A Microfluidic Cell Culture Laboratory

Tissue function is modulated by the spatiotemporal organization of cells and biomolecules on a micrometer scale. In vivo, cells respond to a myriad signaling factors, either in the form of freely-diffusing molecules—e.g. enzymes, nutrients, small ions, growth factors secreted by cells that can be adjacent or as far as several meters for a large mammal—or in the form of relatively immobilized molecules that are anchored to the membrane of adjacent cells (e.g. membrane receptors, cadherins) or bound to extracellular scaffolds, such as the extracellular matrix (ECM) or bone; in addition, cells are also responsive to the physical topography and stiffness of the matrix. All these factors vary locally in smoothly graded or sharp concentration changes at the cellular and sub-cellular scale, as conceptually depicted in Fig. 19.1. This multi-signal changing environment is particularly dynamic during development, wound healing, and cancer.