Un modello epiteliale bronchiale interfaccia aria-liquido per un'esposizione realistica e ripetuta all'inalazione alle particelle trasportate dall'aria per i test di tossicità

This protocol describes how to culture and exposure, human bronchial epithelial cells via the air.This is a more realistic model for studying exposure of the lung to inhaled particles.The lung cells prepared with these methods, to resemble the real environment in the lung.The cells will produce mucus, form a tight barrier, remaining viable for several weeks.This method can be used to study toxic effects of airborne particles.It can also be used to study infectious diseases of the airways and drug taken by inhalation.When attempting this protocol for the first time, keep in mind that generating an agile requires specialist's knowledge.It also requires dedicated equipment for measurement.Begin by diluting the cells in pre-warmed cell culture medium or CCM, to a concentration of 500, 000 cells per milliliter for 6-well inserts.And 250, 000 cells per milliliter for 12-well inserts.Place a cell culture plate with inserts under aseptic conditions, and fill the basal lateral side with pre-warmed CCM.After mixing the cell suspension, pipette it on top of the membrane in the cell culture insert.Cover the plate and incubate it at 37 degrees Celsius and 5%carbon dioxide.Culture the cells under submerged conditions for seven days, to allow them to reach confluency, changing the CCM every two to three days.To measure the transepithelial electrical resistance, use a charged epithelial voltohmmeter, supplemented with a chopstick electrode set.Clean the electrodes with 70%ethanol.Place the longer electrode in the external culture media, until it touches the bottom of the dish.And the shorter electrode in the media, without touching the membrane.Begin collecting measurements on the empty insert without cells.Wait until the measurement stabilizes and write down the resistance in ohms.This measurement is the resistance of the insert membrane without any cells or blank resistance.Repeat the measurement for each insert, and subtract the blank resistance to obtain the true resistance, and multiply by the surface area of the insert.Remove the medium from the apical side of the inserts and add pre-warmed CCM to the basolateral side of the well.The medium should touch the membrane from the bottom, but not leak onto the top of the insert.Culture the cells at the air-liquid interface in the incubator, at 37 degrees Celsius and 5%carbon dioxide for seven days.Changing the basolateral medium every two to three days.Shortly before particle exposure, prepare a 1%nanoparticle suspension.Pre-wet the NPs with 96%ethanol.Add pure water to a final concentration of 1%NPs.Put the vial in a beaker on ice.Sonicate the suspension for 16 minutes, add a stir to the bottle with the dispersion, then connect the bottle to a peristaltic pump via a small tube and adjust the flow to 25 milliliters per hour.One day before exposure, turn on the automated exposure station or AES and allow the cabinet to reach a temperature of 37