Microfluidic studies of apoptosis in heart disease and cancer



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Heart disease and cancer continues to be the leading cause of death. Apoptosis plays a major role in progression of heart disease by contributing to heart muscle loss. On the other hand, apoptosis is inhibited in cancer causing cancer growth. Several microfluidic devices were developed to study apoptosis in heart disease and cancer. Microfluidic devices with oxygen sensing capabilities were fabricated to study cell death during ischemia/reperfusion injury in porcine cardiomyocytes. The functional decline of heart muscle cells has been linked to the development of heart failure. Apoptosis has been identified as a major mechanism of the loss of cardiomyocytes. On-chip oxygen sensing was achieved using oxygen sensitive fluorescent dye to create hypoxic microenvironment for cells. Cardiomyocytes were cultured in a low-shear microfluidic culture chamber at <1% oxygen for 3-5 hours to study cellular response to ischemia. The oxygen supply was subsequently returned to 21% to study cellular response to reperfusion. Mitochondrial depolarization, the earliest stage of apoptosis was studied by assaying mitochondrial membrane potential using the fluorescent probe, MitoTracker Red. We observed that mitochondrial depolarization is initiated in ischemia as well as reperfusion, but the fraction of apoptotic death is larger during reperfusion. The fraction of apoptotic cells were observed to be 3 % at two hours of ischemia and increased up to 22% at the end of ischemia period as compared to 0% in the control samples. Our results were comparable to studies performed on whole animals. Our findings show that primary porcine cardiomyocytes are susceptible to apoptosis after only short periods of ischemia followed by sudden reperfusion.
The microfluidic culture device and oxygen sensing capabilities developed in this work were also modified to study the effects of hypoxia on drug resistance of cancer cells. A vacuum actuated low-shear microfluidic device was used to study the drug response of prostate cancer (PC3) cells and Ramos B cells. Cells were cultured in the chip upto 16 hours and treated with staurosporine to induce apoptosis. The loss of mitochondrial membrane potential was measured using MitoTracker Deep Red; phosphatidylserine externalization was measured using Annexin V coupled to Alexa Fluor 647. For hypoxic samples, the chip was placed in a hypoxia chamber and pre-conditioned at <1% oxygen before inducing the cells with staurosporine. At 1 hour, PC3 cells exposed to 2 µm staurosporine were 32% ± 10% apoptotic under normoxic conditions but only 1.5% ± 12% apoptotic under hypoxic conditions. Whereas, 86% ± 9% of Ramos cells were apoptotic under hypoxic and normoxic conditions, indicating that hypoxia response is more developed in solid tumors when compared with circulating cancer cells. As little as 1 hour of hypoxic preconditioning increased drug resistance in PC3 cells. Our results indicate that hypoxic microenvironments and drug resistance have a strong correlation. Our device was capable of on-chip apoptosis assays, and our approach was able to produce hypoxic environments with facile measurement of oxygen concentration. Finally, to study the changes in protein glycosylation patterns, microfluidic capillary electrophoresis devices with integrated electrospray capability were developed. Glycans have been reported to be involved in several biological processes and the attachment of glycans to proteins during glycosylation can be significantly altered in disease conditions. Multiple glycosylation sites lead to structural variations, which presents a significant challenge in glycan analysis. Capillary electrophoretic techniques provide isomer resolutions but not structural elucidation. At the same time, mass spectrometric techniques lack isomer resolution but provide structural information. Therefore, combining these two techniques enables the separation and identification of complex glycans. Material such as polydimethylsiloxane (PDMS) was used to develop separation and electrospray devices. Dynamic coating of PDMS devices was explored using ionic and non-ionic surfactants. Separation of APTS (8-Aminopyerene-1,3,6-Trisulfonic Acid) labeled glycans were achieved in PDMS devices dynamically coated with cetyltrimethylammonium bromide (CTAB) and dodecyl maltoside (DDM) at 400 v/cm to 520 v/cm. However, when the glycan samples were electrosprayed into a mass spectrometer, the glycan signal was suppressed because of the presence of surfactants. Therefore, alternative material, glass, was explored. Glass devices were fabricated using wet etching and separation and electrospray capabilities were explored. The fabrication of glass devices is relatively time-consuming and difficult as compared with PDMS devices. However, the surface property of glass is much more controllable compared to PDMS. This work encompasses the study of apoptosis in ischemia/reperfusion injury and cancer. Our aim is to provide a better understanding of cell death in heart disease and cancer so that, in future therapies can be developed to block apoptosis in heart disease and to induce apoptosis in cancer.



Microfluidics, Apoptosis, Fluorescence microscopy