Category: Micro- and Nanotechnologies
Ischemic heart disease (IHD) is the leading cause of death in the world. Irreversible cell damages are a major side effect of this disease. Currently, heart transplantation is the most effective method of treatment. However, donor organ shortage and the response of immune system are the main limitations. For this reason, it is necessary to develop alternative methods for treating this ailment. Due to the fact, that Lab-on-a-Chip systems enable to mimic in vivo microenvironment, they have become an appropriate tool used in creating heart disease model. In contrast to other works, here we present a microfluidic system to simulate hypoxia in myocardial tissue using biochemical pathway.
The PDMS/glass microsystem was fabricated using replica moulding techniques. The presented microfluidic system consists of a cell culture microchannel and two side microchannels separated by two rows of pillar. Designed microsystem is matched with specrofluorometric plate reader, which allowed for quantitative fluorescent measurements. Rat cardiomyocytes H9C2 were seeded in the microsystems with a flow rate of 5 µl/min and cultured overnight. Then, hypoxia was induced by mitochondrial oxidative phosphorylation uncoupler - Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). In the experiments, different concentrations of FCCP (10, 75 µM) and incubation time (30, 60, 90, 120 min) were analysed. FCCP was provided to the cells with a flow rate of 1 µl/min. Cell apoptosis was evaluated using cationic carbocyanine dye – tetraethylbenzimidazolylcarbocyanine iodide (JC-1). JC-1 accumulates in the mitochondria of living cells and forms aggregates yields red fluorescence (λem=590 nm). Decline of mitochondrial membrane potential in apoptotic (hypoxic) cells is characterized by disintegration aggregates for monomers and green fluorescent (λem=530 nm). Moreover, effect of FCCP on intracellular calcium ion concentration was analyzed using fluorescent dye – Fluo 4. Quantitative measurements of fluorescence intensity were performed for cells incubated with different concentration of FCCP (10, 75 µM) for 30, 60, 90 and 120 min.
For cells incubated with 10 µM FCCP for 60 min, the decrease of JC-1 aggregates to monomers ratio was noticed. It shows, that cells were in the state of apoptosis. Simultaneously, microscopic observation was performed. It confirmed high number of hypoxic cells (green objects) for this incubation time. This indicates that the selected conditions stimulate hypoxia of cardiac cells in the presented microsystem. Moreover, it was found that concentration of calcium ions increased with FCCP concentration and incubation time.
In this work, we presented Lab-on-a-Chip system for creating a myocardial hypoxia cell model. Additionally, the microsystem is matched with standard plate reader. Moreover, the presented microdevice allows to create model of myocardial infarction and it could be useful in research of alternative methods of IHD treatments and cell therapy based on stem cells.
This work was realized with the frame of project LIDER No. LIDER/026/573/L-4/12/NCBR/2013
Anna Kobuszewska– PhD Student, Warsaw University of Technology, Warsaw, Mazowieckie, Poland
Warsaw University of Technology
Warsaw, Mazowieckie, Poland
I graduated in Biotechnology at Warsaw University of Technology in 2015. I have been a PhD student for two years. Contemporary, I work on creating a heart model after hypoxia in microfluidic system and cell therapy based on stem cells. For last two years I have gained an experience in Lab-on-a-Chip fabrication, working with multiple cell lines and performing cytotoxicity tests. I am co-author of article in SLAS Technology and a chapter in a monograph entitle "Cardiac Cell Culture Technologies - Microfluidic and On-Chip Systems" (Springer). I also participated in numerous international and national conferences (i.e. microTAS).