The overall goal of the following experiment is to observe the effect of chlorine exposure in in vitro cell culture models of the lung, airway and heart. This is achieved by first culturing rat cardiomyocytes or human airway epithelial cells. The rat cardiomyocyte cultures will be exposed to chlorine gas to evaluate the chlorine toxicity against vital organs such as the heart, whereas the exposure of differentiated cultures of human airway, epithelial cells to chlorine allows the further modeling of human exposures to toxic gases.
Ultimately, caspase three seven release and trans epithelial electrical resistance can be measured to determine the apoptotic cell death of the cardiomyocytes and the membrane disruption and cell death of the human airway epithelial cells respectively. The main advantage of this method is that it involves primary tissue cell cultures instead of whole animals, hence allowing a simple in vitro chemical testing method. Tip four, perform chlorine exposure experiments.
The chlorine exposure system must be contained inside a qualified chemical hood with an operational face velocity of 100 FPM before beginning the exposure. First, adjust the system to a slight positive pressure with half an inch of water, then feed dry air and an appropriate level of chlorine into the system at 15 liters per minute through a mass flow controller, the chlorine exposure system uses a compressed gas cylinder containing 1%chlorine in dry nitrogen. Next, lock the chamber lid with the four mini biocon containment unit locks and the low durometer silicone gasket to provide the pressure seal.
And then using the custom designed flow panel, regulate the dilution airflow and the chlorine concentration to the exposure chambers. A low volume sampling pump pulls the exhaust from the chambers into a chlorine analyzer to monitor concentrations that are then recorded on a data logger connected to the analyzer. Use a flow meter to measure the flow rates within the chambers prior to exposure to ensure equal delivery and exhaust rates.
Now, remove the supernatant media from the cells of interest and add fresh media to the cell culture. Then expose the cell cultures to the chlorine gas at the appropriate PPM and exposure period in the two sealed poly cell phone. Biocon containment chambers.
After the exposure, flush the chambers with air for about five minutes until the chlorine level falls below one PPM for safe opening of the system and removal of the cells. To measure the trans epithelial electrical resistance of the chlorine exposed cell cultures, use an epithelial ome meter with a pair of silver chloride chopstick electrodes. First equilibrate the chopstick electrodes in the air liquid interface media 15 minutes before use.
Then add one milliliter of warm media to the apical surface and two milliliters to the basolateral surface of the cell culture. Now dip the shorter arm of the electrode into the apical media and the longer arm into the basolateral media, and click the measure button on the vol meter to evaluate the electrical resistance. Then to determine the trans epithelial electrical resistance, subtract the resistance across a cell-free culture support from the resistance measured across each cell layer.
Primary, raw shaped cardiomyocytes attach, spread, and differentiate on laminin matrices into confluent cultures. In this representative experiment, the RAC cardiomyocytes were further characterized on the basis of their sarcomeric actin and circa two expression rat cardiomyocytes are highly susceptible to chlorine toxicity. A 15 minute exposure to 100 PP M chlorine causes extensive cell rounding and death in submerged cultures, and a disruption of the confluent layers on cells observed on the laminin coated membranes.
The cells also exhibit an enhanced apoptotic cell death as indicated by the release of Caspase three seven in cardiomyocytes grown on inserts. In these next figures, a representative experiment with chlorine exposed, differentiated human airway. Epithelium is shown.
Exposure of human airway epithelium to chlorine causes sloughing and lifting of cell membranes at both low and high concentrations damaged by a low chlorine concentration is quickly reversed. However, in cells exposed to higher chlorine concentrations, the ability to recover from the gas exposure is delayed or absent as observed by visual inspection or cell proliferation assessment by KI 67, staining trans epithelial electrical resistance measurements and caspase activity further confirm these results and provide evidence for a loss of membrane integrity and apoptotic cell death upon chlorine exposure. Thus, these studies describe the development of an in vitro chlorine exposure system that causes loss of membrane integrity and cell death of airway, epithelium and cardiomyocytes After its development.
This technique paved the way for researchers to explore the mechanisms of injury of poisonous gases in complex organs such as the lung and the heart.
