The Differentiating Ability of Rat Lens Epithelial Cells in Cell Culture

Abstract

Dissociated cells of lens epithelia of adult .rats were monolayerly cultured in vitro. After about 15-20 days' period of active cell growth, such characteristic structures that correspond to "lentoid bodies" described previously in chick cultures were formed. These structures consisted of elongated cells, ultrastructural profile of which was similar with lens fiber. The presence of gammacrystallin, a marker molecule specific to mature lens fiber, was confirmed in these elongated cells by means of fluorescent antibody technique. The differentiation of lens fiber in vitro was also recognized in clones originating from single lens epithelial cells cultured at very low cell density.Establishment of such culture systems that permit the differentiation of specific cell types of monolayerly growing cells in vitro is very useful for studying the mechanism of cell differentiation. It will enable us direct observations of the process of differentiation in living culture together with the estimation of growth rate. These culture systems will be beneficial to assay the effect of various biological active substances upon differentiation.In the case of lens differentiation from lens epithelial cells, a number of previous works had failed in obtaining lens differentiation of authentic nature in monolayer cultures (cf. 4, 13), before the success by OKADA et al. using chick lens epithelial cells (5, 6). Extention of similar studies to mammalian materials has not been wholly successful except for the case of the lens epithelial cells of cataract mice (1 2; see 14 for bovine cells). In this paper, we present evidence that lens epithelial cells of adult rat differentiate into characteristic lens fibers in monolayer culture. The differentiation of lens fibers in clones originating from single lens epithelial cells was also demonstrated. MATERIALS AND METHODS Materials for experiments. Methods of culturing lens epithelial cellsEye balls were removed immediately after killing, sterilized by brief immersion (for about 30 sec) in 70% ethanol and washed thoroughly with Hanks' saline. Under a dissecting microscope, lenses were isolated, and lens capsules were peeled off from the center of anterior region toward periphery of lens. The lens epithelium with negligible comtamination of lens fibers attached to capsules. Isolated capsules with underlying epithelia were incubated for 15 min at 37°C in 1 mM EDTA in Ca-and Mgfree Hanks' saline (CMF) and then 0.25% crude trypsin in CMF at 37°C for another 30 min. After removal of trypsin solution as much as possible, the softened tissues were dissociated into single cells by pipetting in the culture medium (see below). The culture medium was mostly Eagles' minimum essential medium with glutamine (E), and Ham' F-12 (F) was also used in some of low density cultures. Both media were supplemented with 10% or 15% fetal calf serum, and they will be designated as E-10, E-15, F-10 and F-15, respectively. All cultures were carried out in Falcon plastic dishes with 6 cm of diameter and kept at 37°C in a humi...

A Study of Human Senile Cataract: Growth and Differentiation of Lens Epithelial Cells in in vitro culture

“…Since the differentiation of the lentoid body from the lens epithelial cell cultured in vitro was shown in chick [15,16], similar studies were achiev ed in rats [8], normal and hereditary cataract mice [20], and in human fe tuses [9].…”

“…The results of immuno chemical examinations can be regarded as the substantial evidence for drawing this conclusion, because the synthesis of y-crystallin occurs pre dominantly in lens fibers in mammalian species [1,8,9,17,18]. Our pre liminary observations also revealed that the lentoids obtained in the present culture were mainly constituted by differentiated cells with the ultrastruc tural characteristics specialized in the fully differentiated lens fiber [8,9,14,17], Such a cell population, however, must be much less in number in com parison with the younger normal lenses, although no comparative analysis has been done in human lenses so far. In relation to this argument, the results of clonal cultures of chick lenses are highly relevant.…”

Crystallins of Lens Epithelial Cells during Aging and Differentiation

“…These results may be compared with the data of Creighton et al [1976] and Hamada and Okada [1977] who also demonstrated the in vitro synthesis of y-crystallin and the formation of lentoid bodies in systems of rat lens epithelial cells.…”

The Influence of Culture Conditions on the Behaviour of Lens Epithelial Cells

“…Whereas M 199 favours the formation of elongated cell forms, MEM cultured cells exhibit an epitheliallike morphology and lead to dense aggregates. Most authors describing the formation of lentoid bodies used this type of medium [6,11], Araki and Okada [1] could demonstrate that also the process of foreign differentiation of neural retina cells depends on the choice of medium. Additionally to these morphological aspects the growth rate of cultured calf lens epithelial cells is also affected.…”

“…With respect to rat lens epithelial cells. Hamada and Okada [6] described the formation of lens-like structurescalled lentoid bodies. Creighton et al [5] also observed lentoid bodies and additionally described a remarkable fiber-like elongation of the former pave ment-like cells.…”