Morphology of cell growths of fetal lens organ cultures

Abstract

When the integrity of the fetal lens in tissue culture is disturbed it undergoes dedifferentiation and incomplete differentiation of the nucleated cells of the lens. Cell growths which form under these circumstances resemble the cellular masses of the lens affected by cataracts [1-3]. The question arises: what is the role of integrity of thelenticular capsule and of the lens itself in the genesis of these abnormal cell growths. Existing information on organ cultures of the lens relates to short-term (1-3 days) cultures of adult animals, used for histophysiologic studies, and no morphological data are available [4]. There are likewise no data in the literature on more prolonged culture of mamalian fetal lenses. The investigation described below was carried out in order to fill this gap. EXPERIMENTAL METHOD Altogether 144 lenses from 72 pig fetuses 10-20 cm long were cultured in penicillin flasks closed with rubber stoppers. Eagle's or DMEM medium with the addition of 6% or 20% fetal calf serum was used. Penicillin and streptomycin were added to all media in a dose of I00 U/ml to prevent bacterial contamination. A lens was placed in each flask, covered with 3 ml of nutrient medium, and incubated at 37~ The medium was renewed on the 2nd day of culture and thereafter every 2 or 3 days, with replacement of 3 and 4 ml throughout the period of culture (30-42 days). The lenses were studied daily with the naked eye to detect any opacity. Material for histological study was fixed on the 2nd-3rd and 6th-7th days, and thereafter at intervals of 1 week until the end of the experiment. Fixation was carried out in Carnoy's and Bouin's fluid or in a 10% solution of formalin. Sections 7-10 ~ thick were stained with hematoxylin and eosin. Areas for electron microscopy were chosen under a binocular loupe, cut out, and treated by the usual method for electron microscopy. Very thin sections from the same block of epithelioid and fibroblast-like cells and from five blocks of vesicular cells were studied in the EVM 100BR microscope. EXPERIMENTAL RESULTS Original normal fetal lenses for culture were characterized by a simple cubical anterior epithelium, which change on the side of the equator into pseudostratified epithelium in the germinative zone. The cells were then arranged in meridional rows and later formed adult lens fibers (Fig. i). After culture for 24 h slight cortical opacity was observed in the zone of the anterior epithelium, and this partly disappeared by the 3rd day in culture. Throughout the period of culture speckled cortical opacities persisted, gradually widening and producing total opacity of the lens after 30-40 days of culture. Under the light microscope, opacities at the beginning of culture corresponded to cortical lacunae, containing fluid, between the cortical lens fibers. Later the opacities were due not only to lacunae, but also to cell growth starting to be formed at that time, in conjunction with lacunae. In Eagle's medium lacunae were found mainly in the equatorial part of the lens, whereas in DMEM medium they were most common in the posterior cortex. Cells of the anterior epithelium of the lens in culture remained in a single layer, except in certain areas in which, starting with the 3rd day of Culture, agglomerations of epithelioid and fibroblast-like cells appeared between the capsule and lens fibers, together with infiltrating cells migrating from the anterior epithelium and insinuating themselves , m