Mycoplasma pneumoniae infection of intact guinea pig tracheas cultured in a unique matrix-embed/perfusion system

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Abstract
A new method for the in vitro culture of entire, intact tracheas from adult guinea pigs is described. Matrix-embed/perfusion (MEP) culture is based on an immobilization of the tissue in nutrient agar. The tubular piece of agar-embedded organ was contained in a special perfusion block with two wells for liquid medium at either end. When incubated on a rocker platform, liquid medium flows through the trachea and supplies oxygen and nutrients. In this configuration, tracheas maintain near-normal metabolism (ATP content and dehydrogenase activity), structure (as determined by light and electron microscopy), and function (ciliary motion). Tissues could be maintained in vitro in a normal state for at least 4 wk, with reduced ciliary motion and cell metabolism detectable for at least 6 wk. Agarembedded tissues from the MEP cultures were nearly identical to those cultivated with standard tracheal ring explant techniques. Tracheas in the MEP cultures were infected withMycoplasma pneumoniae. Attachment was neuraminidase-sensitive. Mycoplasma attachment was lowest on the epithelium along the dorsal ridge, but was uniform along the length of the trachea. Ciliostasis and cytonecrosis induced byM. pneumoniae was dose dependent. The matrix-embed/perfuse technique appears to have considerable potential for several types of in vitro studies on trachea or other tubular organs.