Ischemia/reperfusion (I/R) injury was induced in primary porcine cardiomyocytes in a low-shear microfluidic culture chip. The chip was capable of sustaining cardiomyocyte culture and inducing I/R injury by subjecting the cells to periods of hypoxia lasting 3-4 hours followed by normoxia. Mitochondrial membrane potential was assayed using MitoTracker Red to follow mitochondrial depolarization, the earliest stage of apoptosis. Cell adhesion and morphology were also determined simultaneously with fluorescence measurements. Changes in membrane potential were observed earlier than previously reported, with mitochondria becoming depolarized as early as 2 hours into the ischemia period. The cells with depolarized mitochondria were deemed apoptotic. Out of 38-61 cells per time frame, the fraction of apoptotic cells was found to be similar to control samples (3%) at two hours of ischemia, which increased up to 22% at the end of ischemia period as compared to 0% in the control samples. Morphological analysis of cells showed that 4 hours of ischemia followed by reperfusion produced blebbing cells within 2 hours of restoring oxygen to the chip. This approach is a versatile method for cardiomyocyte stress, and in future work additional analytical probes can be incorporated for a multi-analyte assays of cardiomyocyte apoptosis.

A microfluidic system for cell culture and drug response studies was developed to elucidate the effects of hypoxia on drug susceptibility. Drug response studies were performed in prostate cancer cells and Ramos B cells under normoxic and hypoxic conditions. A vacuum actuated microfluidic culture device was used for cell culture and PC3 cells were cultured in the chip up to 16 hours. Cells were treated with several concentrations of staurosporine and apoptosis was assayed using the fluorescent probes MitoTracker Red and Annexin-V. For hypoxic samples, the chip was placed in a hypoxia chamber and pre-conditioned at <1% oxygen before inducing the cells with staurosporine. Cells exposed to 2 μM staurosporine were 32% ± 10% apoptotic under normoxic conditions but only 1.5% ± 12% apoptotic under hypoxic conditions. As little as 1 hour of hypoxic preconditioning increased drug resistance. Cell apoptosis correlated with drug dose, although in each case hypoxia reduced the apoptotic fraction significantly. Given the rapid nature of cell adaptation to hypoxia, this chip and analysis approach can be used to identify compounds that can induce cell death in hypoxic tumor cells rapidly.

Background and Objectives Infusion of by-products of red blood cell (RBC) storage-induced degradation as well as of the residual plasma proteins and the anticoagulant-preservative solution contained in units of stored blood serve no therapeutic purpose and may be harmful to some patients. Here, we describe a prototype of a gravity-driven system for bedside washing of stored RBCs.Materials and Methods Stored RBCs were diluted to 10% haematocrit (Hct) with normal saline, matching the conventional washing procedure. The dilute RBC suspensions were passed through a column of coiled tubing to allow RBC sedimentation in normal gravity, thus separating them from the washing solution. Washed RBCs were collected using bifurcations located along the tubing. Washing efficiency was quantified by measuring Hct, morphology, deformability, free haemoglobin and total-free protein.Results The gravity-driven washing system operating at 0Á5 ml/min produced washed RBCs with final Hct of 36Á7 -3Á4% (32Á3-41Á2%, n = 10) and waste Hct of 3Á4 -0Á7% (2Á4-4Á3%, n = 10), while removing 80% of free haemoglobin and 90% of total-free protein. Washing improved the ability of stored RBCs to perfuse an artificial microvascular network by 20%. The efficiency of washing performed using the gravity-driven system was not significantly different than that of conventional centrifugation.Conclusions This proof-of-concept study demonstrates the feasibility of washing stored RBCs using a simple, disposable system with efficiency comparable to that of conventional centrifugation, and thus represents a significant first step towards enabling low-cost washing of stored blood at bedside.

Apoptosis plays a major role in both healthy and diseased cells. The analysis of apoptosis can take advantage of multiple cellular markers, enabling the process to be studied at different time points. In this chapter, several apoptosis assay protocols are provided.

Several analytical methods, many of which rely on fluorescence processes, have been developed to study apoptosis (programmed cell death). Apoptosis is a highly regulated biological event and is a vital process that helps regulate tissue growth, normal cell turnover, immune response, and tissue development. However, diseases such as cancer and heart disease are associated with malfunctions in the apoptosis machinery. There is therefore a need to elucidate the processes of apoptosis induction and inhibition. Fluorescence assays continue to play a major role in apoptosis assays, and developments in probe and fluorescent methods for assaying apoptosis are ongoing. There are several standard techniques, such as flow cytometry and confocal microscopy, for apoptosis study in cells. In addition, new techniques such as superresolution microscopy, multiphoton excitation, and single‐cell−single‐molecule spectroscopy are quickly emerging. This article explores several fluorescence approaches used in apoptosis studies as well as describes the mechanisms and hallmarks of the apoptotic cascade. As apoptosis plays a very important role in both healthy and diseased organism functions, the need to develop and apply sensitive analytical methods continues to be of the utmost importance.

Several analytical methods have been developed to study apoptosis (programmed cell death), many of which rely on fluorescence processes. Apoptosis is a highly regulated biological event and is a vital process that helps regulate tissue growth, normal cell turnover, immune response, and tissue development. However, diseases such as cancer and heart disease are associated with malfunctions in the apoptosis machinery. There is, therefore, a need to elucidate the processes of apoptosis induction and inhibition. Fluorescence assays continue to play a major role in apoptosis assays, and probe and method development are ongoing. There are several standard techniques such as flow cytometry and confocal microscopy for apoptosis study in cells. In addition, new techniques such as super‐resolution microscopy, multiphoton excitation, and single cell‐single molecule spectroscopy are quickly emerging. This updated article will explore several fluorescence approaches used in apoptosis studies as well as describe the mechanisms and hallmarks of the apoptosis cascade. Since apoptosis plays such an important role in both healthy and diseased organism function, the need to develop and apply sensitive analytical methods continues to be of the utmost importance.

Background and aimsExtracellular vesicles (EVs) play an important role in maintaining endothelial cell (EC) homeostasis. Here, we examined the effects of exosomes and microvesicles, two main subtypes of EVs, derived from either ECs or macrophages on endothelial inflammation.MethodsExosomes and microvesicles were isolated from the cell culture medium from mouse microvascular ECs and J774 macrophages by sequential ultracentrifugation. Exosomes were detected by Western blotting analysis of marker protein CD63 and AIP‐1. Microvesicles were stained with FITC‐annexin V and Hoechst 33342 and analyzed by flow cytometry. Endothelial inflammation was examined by analyzing mRNA and protein expression of VCAM‐1 and monocyte adhesion using real time RT‐PCR and fluorescence microscopy.Result and conclusionBoth macrophages and ECs spontaneously released CD63 and AIP‐1 positive exosomes under resting condition, whereas the expressions of these exosome markers were not affected by TNFα stimulation. In contrast, TNFα markedly increased the release of annexin V+ microvesicles from either ECs or macrophages. Interestingly, among these microvesicles, we identified a novel subpopulation of microvesicles that are positively stained with both annexin V and Hoechst 33342. This result suggests that TNFα increased the release of a unique subset of microvesicles containing DNA fragments. Functionally, EVs (microvesicles and exosomes) derived from TNFα‐treated macrophages, but not from TNFα‐treated ECs, increased VCAM‐1 expression and monocyte adhesion in naïve ECs. Together, these results suggest that EVs may exert distinct effects (pro‐inflammatory v.s. anti‐inflammatory) on endothelial homeostasis depending on their subtypes and/or cellular sources.Support or Funding InformationSupported by NIH grants HL122769 and HL122937