Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and endothelial cells, provide an ideal platform for an updated status on the immune condition of an individual. The microfluidic protocol allows for enrichment of CNCs without selective cell loss and sample preparation variability due to user-mediated steps. Briefly, the protocol includes device fabrication, sample collection, device setup, and running blood through the microfluidic chamber. Within the device whole blood is rapidly mixed with deionized water for approximately 10 seconds in a 50 micron x 150 micron microfluidic channel. In this time span erythrocytes are lysed due to hypotonic conditions. Herringbone structures on the bottom of the channel ensure thorough mixing and exposure of cells to a constant environment. Remaining cells are returned to isotonic conditions at the exit of the device, fixed using 2% paraformaldehyde, centrifuged to separate erythrocyte debris from CNCs, and suspended in flow buffer for staining and analysis by flow cytometry. Results show clean flow cytometry scatter plots with CNC populations saved. Significance of this device and protocol comes in the study and understanding of disease pathogenesis by analysis of CNC populations. Hence, automation, effectiveness, and simplicity of the microfluidic protocol are demonstrated

Plastic based microfluidic systems and their applications in biology.

This research work focuses on the development of plastic microfabrication techniques, specifically epoxy based casting for the fabrication of microfluidic platforms that can then be used for different chemical and biological applications. Over the past few years there have been various applications of miniaturization in life sciences for different areas like drug discovery in the pharmaceutical industry, molecular recognition in clinical diagnostics, cell culture and manipulation for cellular and tissue engineering, which have radically changed the way in which information is processed and experiments are performed. A casting technique using optical grade epoxies has been developed to fabricate these microfluidic systems. Miniaturization offers the possibility to integrate multiple functions onto a single platform. Two separate techniques for integration of control elements onto plastic based systems are discussed. The first one involves embedding active silicon micromachined devices in plastic microsystems using a Polymer Flip chip process and the other involves surface micromachining to build from the bottom up devices that can be integrated within the system. A polyethylene glycol (PEG) based actuator has been developed and used to fabricate nozzle-diffuser pumps that can be integrated easily within a microfluidic system. Flow rates of up to 80 nl/min and pressures of up to 1400 Pa can be generated. Work has been done in developing tools for molecular and cellular biology applications. Fabricated devices can be used for molecular assays of bio-molecules like nucleic acids and proteins. Demonstration devices were designed and fabricated to perform Polymerase Chain Reaction (PCR) and Capillary electrophoresis (CE). Also, application of microfluidics and microfabrication can be used to engineer cellular interactions with surfaces and surroundings. Cell attachment is critical to the normal functioning of the cell and requires Extra Cellular Matrix (ECM) proteins for proper attachment. An electrochemical deposition technique for patterning conductive biomolecules doped with proteins is explained. Laminar flows in channels are used to precisely control the flow of the electrolyte over the electrodes to define the area of deposition. Using this technique precisely controlled deposition of polypyyrole (PPY) doped with collagen is achieved on gold microelectrodes.Ph.D.Applied SciencesBiomedical engineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/123274/2/3068959.pd

Microfluidic Adaptation of Density-Gradient Centrifugation for Isolation of Particles and Cells

Density-gradient centrifugation is a label-free approach that has been extensively used for cell separations. Though elegant, this process is time-consuming (>30 min), subjects cells to high levels of stress (>350 g) and relies on user skill to enable fractionation of cells that layer as a narrow band between the density-gradient medium and platelet-rich plasma. We hypothesized that microfluidic adaptation of this technique could transform this process into a rapid fractionation approach where samples are separated in a continuous fashion while being exposed to lower levels of stress (<100 g) for shorter durations of time (<3 min). To demonstrate proof-of-concept, we designed a microfluidic density-gradient centrifugation device and constructed a setup to introduce samples and medium like Ficoll in a continuous, pump-less fashion where cells and particles can be exposed to centrifugal force and separated via different outlets. Proof-of-concept studies using binary mixtures of low-density polystyrene beads (1.02 g/cm3) and high-density silicon dioxide beads (2.2 g/cm3) with Ficoll–Paque (1.06 g/cm3) show that separation is indeed feasible with >99% separation efficiency suggesting that this approach can be further adapted for separation of cells

Tissue Chips and Microphysiological Systems for Disease Modeling and Drug Testing

Tissue chips (TCs) and microphysiological systems (MPSs) that incorporate human cells are novel platforms to model disease and screen drugs and provide an alternative to traditional animal studies. This review highlights the basic definitions of TCs and MPSs, examines four major organs/tissues, identifies critical parameters for organization and function (tissue organization, blood flow, and physical stresses), reviews current microfluidic approaches to recreate tissues, and discusses current shortcomings and future directions for the development and application of these technologies. The organs emphasized are those involved in the metabolism or excretion of drugs (hepatic and renal systems) and organs sensitive to drug toxicity (cardiovascular system). This article examines the microfluidic/microfabrication approaches for each organ individually and identifies specific examples of TCs. This review will provide an excellent starting point for understanding, designing, and constructing novel TCs for possible integration within MPS

Tissue Chips and Microphysiological Systems for Disease Modeling and Drug Testing

Tissue chips (TCs) and microphysiological systems (MPSs) that incorporate human cells are novel platforms to model disease and screen drugs and provide an alternative to traditional animal studies. This review highlights the basic definitions of TCs and MPSs, examines four major organs/tissues, identifies critical parameters for organization and function (tissue organization, blood flow, and physical stresses), reviews current microfluidic approaches to recreate tissues, and discusses current shortcomings and future directions for the development and application of these technologies. The organs emphasized are those involved in the metabolism or excretion of drugs (hepatic and renal systems) and organs sensitive to drug toxicity (cardiovascular system). This article examines the microfluidic/microfabrication approaches for each organ individually and identifies specific examples of TCs. This review will provide an excellent starting point for understanding, designing, and constructing novel TCs for possible integration within MPS

Biomimetic Cardiac Tissue Model Enables the Adaption of Human Induced Pluripotent Stem Cell Cardiomyocytes to Physiological Hemodynamic Loads

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) provide a human source of cardiomyocytes for use in cardiovascular research and regenerative medicine. However, attempts to use these cells <i>in vivo</i> have resulted in drastic cell death caused by mechanical, metabolic, and/or exogenous factors. To explore this issue, we designed a Biomimetic Cardiac Tissue Model (BCTM) where various parameters associated with heart function including heart rate, peak-systolic pressure, end-diastolic pressure and volume, end-systolic pressure and volume, and ratio of systole to diastole can all be precisely manipulated to apply hemodynamic loading to culture cells. Using the BCTM, two causes of low survivability in current cardiac stem cell therapies, mechanical and metabolic, were explored. iPSC-CMs were subject to physiologically relevant mechanical loading (50 mmHg systolic, 10% biaxial stretch) in either a low- or high-serum environment and mechanical loads were applied either immediately or gradually. Results confirm that iPSC-CMs subject to mechanical loading in low-serum conditions experienced widespread cell death. The rate of application of stress also played an important role in adaptability to mechanical loading. Under high-serum conditions, iPSC-CMs subject to gradual imposition of stress were comparable to iPSC-CMs maintained in static culture when evaluated in terms of cell viability, sarcomeric structure, action potentials and conduction velocities. In contrast, iPSC-CMs that were immediately exposed to mechanical loading had significantly lower cell viability, destruction of sarcomeres, smaller action potentials, and lower conduction velocities. We report that iPSC-CMs survival under physiologically relevant hemodynamic stress requires gradual imposition of mechanical loads in a nutrient-rich environment