Exp. Eye Res. (1991) 52, 113-119
Human GEORG
Lens
BERMBACH,
Epithelial URSULA
University
Eye Clinic
(Received
1 March
MAYER
Erlangen1989
Cells
Niirnberg,
and accepted
AND
in Tissue
Culture
GOTTFRIED
0. H. NAUMANN
D-85200 in revised
Erlangen, form
Germany
72 April
1990)
In 94 cultures of human cataractouslensepitheiial cellsderived from patientsof different age,the cell diameterand morphology were observedand photographedafter 21 days. The following resultswere obtained: every culture had its typical pattern dependingon the ageof the patient. which was from 3 monthsto 94 yr. The cell culture taken from a young man was characterizedby smallerepitheloidcells showing a distinctly visible nucleus: the homogeneityof the monolayer diminishedin parallel to the increasing age of the patients. The cell morphology becameincreasingly inhomogenous,showed polymorphismand signsof vacuolic degeneration,and averagecell diameterincreasedin parallel with the increasingage.The cellsshowedthree characteristicphenotypes(Fig. 9), the morphologydepending significantly on the age of the culture and of the donor: small epithelial-likecellswithout vacuoles showing a distinctly visible nucleus (most of these had a diameter of SO-100pm), large cells with vacuoles,and cellswithout a visiblenucleusand in rare casesincluding vacuoles(they appearedround and irregular, their diameterwas 200-300 pm, and they included filamentousstructures). Longterm cultureswere maintainedfor 8 monthsand subculturesmaintainedup to the third passage. Key words:human lens: lensepithelialcells: lens: human lensepithelialcells: cataract; cell culture: tissueculture.
1. Introduction The lens derives from ectoderm and consists only of epithelial cells and their products. It is completely separated from all other types of cells by the basement membrane, which acts as a boundary layer. Therefore, lens epithelia can be cultivated without interruption of any nerval or vascular supply and without contamination from other types of cells like fibroblasts (Mayer, 1969 ; Reddan and McGee, 1979). For this reason, cells of such cultures are exclusively epithelial cells. Due to these anatomical facts, the lens is particularly suitable as a model for researching different scientific questions such as growth, mitosis and differentiation as well as protein synthesis and electrolyte transport (Mayer, 1969, 1974; Koch, 1979; Platsch und Wiederholt, 1981). Koch (19 79) described the importance of theoretical, as apposedto functional, basic research of the lens, especially concerning clinical ophthalmology. Cataracts are the most common diseaseof the lens and one of the most frequent causes of blindness (Naumann, 1980). The pathological mechanisms causing the most frequent types of cataract, especially the senile cataract, remains unknown. However, the function of lens epithelia is often discussedin connection with the pathogenesis of cataract (von Sallmann, 19 52, 19 5 7 ; Cogan, Donaldson and Reese, 1952; von Sallmann et al., 1955; Worgul and Rothstein, 19 75; Streeten and Eshagian, 19 78). The purpose of the present studies was to cultivate human lens epithelial cells obtained from surgically removed cataracts and to determine whether morpho00144835/91/020113+07
$03.00/O
logical changes in culture depend on the donors age and/or number of subcultures. The cell cultures system used was basedon that described by Mayer in 1969. This method has been used successfully to culture tissue removed from patients aged 3 months to 94 yr. The question was: do morphological alterations of the cells depend on the donor’s age or on the number of subcultures, and can parallels be found between both ? 2. Materials and Methods The cultures of human lens epithelial cells were all derived from cataractous eyes. The patients were operated on in the University Eye Clinic of ErlangenNiirnberg (Professor G. 0. H. Naumann), and the cataractous lens was removed by extracapsular extraction, i.e. the whole anterior capsule with the adhering subcapsular layer of epithelial cells was cut and removed from the lens and could be cultivated in one piece. The human lens capsules were set as explants in single cultures, with 94 samples, which grew out in culture, being examined. The graph shows the distribution of the age of those patients, whose cells grew out in vitro (Fig. 1). The precondition for the outgrowth of the cells was the attachment of the capsule on the flask bottom. The capsule was spread out with capsule forceps with the cell layer upwards on the surface of a plastic culture flask (Falcon cell culture flask, 50 ml volume, 2 5 cm2, Becton & Dickinson, Heidelberg). Medium was added without touching the capsule. In order to do this, the first flask was set on its end in the incubator 0 1991 AcademicPressLimited BER 52
G. BERMBACH
culture
ET AL.
flask
medium
FIG. 2. Plastic culture flask containing adhering lens epithelial cells.
the capsule with
Age (yr)
FIG. 1. Distribution
of the ages of donor patients.
so that the medium was located under the prepared capsule. In this way the tearing off of the capsule from the surface could be avoided. The rising water vapour protected the tissue from drying, forming a wet chamber. After 1 hr the flask was laid down, so.that the medium covered the epithelium, which was now firmly attached to the bottom (Fig. 2). TCM 199 [Hank’s (1 x ) by Biochrom KG, Berlin] was used as medium for all cultures, with 20% foetal calf serum and specific supplements being added: 2rnM L-glutamine, 100 I.U. penicillin ml-‘, 100 g streptomycin ml-l, 20 mM NaHCO,, 18 mM Hepesbuffer. For subcultivating, the confluent monolayer of the culture was incubated with trypsin and the dissociated cells were split 1: 2, as described by Paul (198 1). The growth of the cultures and the morphology of the cells were studied by an inverted microscope (Leitz) with a phase contrast and an incorporated camera. Kodak Ektachrome SO Asa, having a sensitivity of 18 DIN, was used: Twenty-one days after inoculation, the cells of each culture were photographed and compared by means of biomicroscopic photos. To identify special phenotypes and diameters of cells, the photos and a photographed scalewere projected on the samescreen (Fig. 3). Statistical Procedure To determine whether the diameters of human lens epithelial cells increases with donor age, the largest diameters of individual cultured cells were measured, using sample size of 30-50 cells. The mean and standard deviation for each patient were determined, and Spearman’s rank correlation-coefficient was calculated with respect to age. To ascertain whether pathological cellsappear more
FIG. 3. Projection of the biomicroscope photographed scale to the same screen.
photo
and a
often in cultures of material removed from older patients, the frequency of the three different phenotypes was determined. The number of different phenotypes of cells was set in relation to the patient’s age. Again, using the rank-correlation by Spearman. the 9 5 % confidence interval was calculated and a test for correlation was done. 3. Results Descriptionof Individual Cultures After a 21-day Incubation, Typical Aspects (a) Cellsof a 3-month-old baby [Fig. 4 (A)]. The cells shown in Fig. 1 describe a more rapid and abundant outgrowth than in cultures obtained from older patients. An almost homogenous cell picture could be seenwith the cellsbeing rather small and regular, and the nucleus being distinctly visible in nearly all cells. Their shape was epithelial or polygonal, and their size did not exceed 100 pm on average. The nucleus was relatively large in contrast to the smaller cytoplasm surrounding it, meaning that the nucleocytoplasmic ratio had shifted towards the nucleus. The cells were generally well demarcated from each other, so that we could comment on the membrane. The membrane did not look smooth, but slightly lobulated. At that time neither vacuoles nor granules had appeared in the plasma. (b) Cells of a 42-yr-old man [Fig. 4 (B)]. The elements were well demarcated from each other, and were bigger than those previously demonstrated, reaching a size of 150 pm, but were still uniform. The shape was epitheloid and regular, the nucleus was
HUMAN
LENS
EPITHELIAL
CELLS
FIG. 4. A, Cells of a 3-month-old
FIG. 5. A, Cells of a 62-yr-old
115
baby, x 400. B, Cells of a 42-yr-old
man, x 400. B, Cells of a 73-yr-old
man, x 400.
man, x 400.
mostly visible, and the homogenous cell picture still existed. There were only a few cells containing vacuoles.
cellswithout a distinctly visible nucleus, often showing filamentous structures. There were alsosomeepithelial cells, which were small and regular.
(c) Cells of a 62-yr-oZd mun [Fig. 5 (A)]. Now there was an heterogenous cell picture with pathological cells dominating: they contained vacuoles and were enlarged and irregularly shaped. The pathological cells had a diameter of 200-300 pm and were large
(d) Cells of a 73-yr-oZd man [Fig. 5 (B)]. In this culture, there was no uniform type of cell. The mixed population included apparently young appearing epithelial cells, although these were somewhat larger than their normal counterparts and possesseda large 8-2
116
G. BERMBACH
ET AL.
FIG. 6. A, Cellsof a 94-yr-old woman. x 400. B, Third subculture: 69-yr-old man, 8 months in culture, x 400.
nucleus. Enlarged cells containing cytoplasmatic vacuoles were also observed. In thesecells, the nucleus was rarely visible. The pathologic cells were large, heterogenous, irregularly shaped and were transparent with cytoplasm accumulating at the periphery. (5) Cellsof a 94-yr-oZd woman [Fig. 6 (A)]. Growth capacity was found even in the cells of this 94-yr-old woman. In the centre there was a typically vacuolated cell, surrounded by someenlarged senilecells, without a distinctly visible nucleus. Moreover, some smaller epithelial shaped cells were observed. As already shown in the cells of the above-mentioned patient, the cell picture showed inhomogeneity and could be classifiedin the old-age group. Gapswere found in the layer, which means a confluent monolayer had not been formed. Signs of vacuolic degeneration were predominant. Morphological Descriptionof the Passages Cellular growth extending from the explants occurred after 2-5 days, regardlessof the patient’s age. Initially they were similar to fibroblasts but later they lay together like epithelial cells. forming a monolayer. Mostly the cells formed a confluent layer after 3 weeks, also independent of the patient’s age. With increasing time in culture, signs of cell ageing increased much more rapidly in cells of older patients than in those of younger ones. After only 5 weeks many large and bulgy cellswith filamentous structures could be found in samples of older patients. After 5 months the above-mentioned large transparent cells with smooth membrane and filamentous structures were predominant in the cell picture. Where the
nucleus was still visible, it was surrounded by many vacuoles. In this obviously stationary phase the samplesremained in culture for 8 months. Third subculture [Fig. 6 (B)]. In the third subculture only large senile cells could be found, which were transparent and rigid. The proliferation capacity had apparently stopped and further subculture could not be established. The cells remained in this stationary phase for several months. The morphological appearance of the cell cultures depended on the age of the donor patient. Cultures of material obtained from patients of 3 months to 30 yr of age were homogenous, possesseda minimum number of pathological cells and had an average cell diameter not exceeding 100 pm. Cultures of material removed from patients between 30 and 55 yr of age were still homogenous. but possessedZO-50% pathological cells, and the average cell diameter in these cultures was greater, at 150 pm. Heterogenous cultures were observed when donor material from patients aged 50-65 yr was used: pathological cells dominated and the average cell diameter was 1SO-250 pm. Finally, donors beyond 65 yr of age yielded cultures consisting predominantly of pathological cells (90%). Cell diameters ranged between 100 and 400 ;ern and the cultures were extremely heterogenous. Statistical Evaluation First question: Doesthe cell diameterincreasewith the ageof the donor? We measured the longest diameter of the smallest and of the largest cell in every culture. Having done this, the mean cell diameter of 30-50
HUMAN
350
LENS
EPITHELIAL
CELLS
117
-
x x x x x
x
x $?ix
xdXx xx! ,“x I
30
40
50
/
Ld’” x-x 2~x.x ’ xx ;x x
x
x
x
xx I
I
I
I
60
70
80
90
I
Age group
Age (yr)
FIG. 7. Correlation of donor patient.
between average cell diameter and age
Spear-man’srank correlation coefjcient Spearman correlationscoefficient
Confidenceinterval (95%)
0.516 0.788
0.290-0611 0664-0837
-0.754
of phenotypes
in cor-
TABLE II
TABLE I
Range Mean cellaverage Phenotype 1 (relative frequency) Phenotype 2 (relative frequency) Phenotype 3 (relative frequency)
FIG. 9. Mean relative frequency relation to donor patient’s age.
- 0.604-(
Correlation coefficient, 95 % interval, and P-value of the different phenotypes Age group (Yd O-30
- 0.804) 31-55
0.411
0188-0.598
O-674
0~530-0-597
56-65 > 65
Phenotype 1 2 3 1 2 3 1 2 3 1 2 3
n
6 15 16 57
Mean relative frequency
S.D.
0.873 0.111 0.016 0.656 0.185 0.159 0.434 0.189 0.377 0.166 0.2 70 0.564
0.101 0.101 0028 0.097 0.08 5 0.062 0.158 0090 0.186 0.163 0.137 0.177
P < 0.01.
CELLS WITH DISTINCTLY NUCLEUS; SMALLER; EPITHELIAL MORPHOLOGY
CELLS
WITH
CELLS OFTEN
WITHOUT VISIBLE WITH FILAMENTDUS
FIG.
Secondquestion: Dopathologicalcellsappearmoreoften in cultures taken from older or younger patients? We counted the number of the phenotypes 1, 2 and 3 in every culture (Fig. 8). In each age group, the mean relative frequency (Fig. 9) and the corresponding standard deviation was calculated. The number of phenotype 1 decreasedwith the donor’s age, whereas frequence of phenotypes 2 and 3 increased. In order to prove this, the correlations-coefficient (rank-correlation by Spearman), the 9 5 % confidence-interval and the P-value were calculated. Significant differences in the number of different phenotypes of cells depending on the age of the donor patient were found (Table II).
VISIBLE
VACUOLES
8. Characteristic
NUCLEUS; STRUCTURES
phenotypes
of cells.
4. Discussion cells and the range, that means the variation of the cell diameter in this sample, were calculated. We found that the mean cell diameter did increase with the age of the patient (Fig. 7). Spearman’s rank correlationcoefficient was calculated (Table I) which confirmed that there was a significant (P < 0.01) correlation between the average cell diameter and the age of the donor patient.
The cell culture technique was tist described in 1969 by Mayer who examined the oxygen uptake of cultured bovine lens epithelia. By Warburg’s respirometer she determined the respiratory quotient of the lens epithelium to be of 1.04 (1971). By taking photographs every 30 min for 2 days, Mayer (19 74) found a middle division range of cell division of 10.5 hr. Mayer and Sames (198 1) examined oxygen
G. BERMBACH
118
consumption and ageing in bovine lens epithelia and observed that the oxygen consummation was without any alteration up to the 40th passage. These were our parameters of cell growth. All photos were taken after a 21-day culture, and growth conditions were the same in all cases. It is difficult to perform comparative studies using relatively normal tissue. Clear lens tissue is never surgically removed and donor eyes require extended transport-times and hence the tissue is difficult to culture successfully. The modified method for cultivating human lens epithelial cells described in this paper makes an application for tissue culture possible. which is practicable in a large number and reproduces well. Very little comparable literature can be found on tissue culture of human lens epithelial cells as observed by Ringens et al. (1982). He cites Hamada and Okada (1978). Tassin, Malaise and Courtois (19 79). Eguchi and Kodama (1979) and Perry, Tassin and Courtois (19 79). FranGois and Victoria-Troncoso ( 19 79) are, however, not cited. Only Reddan et al. (1982/83) can be found in recent literature. However, all these authors obtained the operated cataract lenses by cryoextraction. Using this method always destroys a part of the epithelia. In extracapsular cataract extraction the total microsurgical removal of the lens capsule with the adhering subcapsular layer of epithelia is a much more adequate technique. The often used enzymatic dissociation of the epithelial cells from the capsule by trypsine (Hamada and Okada. 1978; Eguchi and Kodama, 1979) damages the cells (Paul, 198 1) and was not successful in our own experiments. The cells need time to recover, but they can also be permanently impaired. Franqois et al. (1979) described a very costly method, which involved sticking the capsule on the flask bottom, but this did not prove successful in our own experiments. Reddan et al. (1982/83) examined the morphology of the cells in culture in eight different media with various content and growth factors: according to his study, human lens cells obtained from normal and cataract lenses showed a similar appearance. However, they had a limited growth capacity in vitro, in spite of the enrichment of the medium by well-known mitogens. Therefore, Reddan came to the conclusion that the present media were still lacking in some essential substrates for the long-term culture of human lens cells. We conclude that lens cells cannot be cultivated as abundantly as tumour cells could be. Such cultures could help in the understanding of factors controlling growth, development, repair and ageing of these cells, and would also make it possible to prove genetic defects connected with congenital cataracts. Ringens et al. (1982) cultivated human foetal lens cells, which also showed limited growth capacity in vitro. He described signs of cell ageing which appeared after 3 months in the primary culture as well as in the
ET AL.
subculture, and also observed vacuolic degeneration and filamentous structures, similar to the observations described above. He did not measure any cell diameter : neither did Hamada and Okada (19 78), Eguchi and Kodama (1979) and Paul (1981). Perry et al. ( 19 79 succeeded in incubating foetal and adult human lens cells, and observed a heterogenous cell picture after 10 weeks, the cultures consisting of young appearing cells, and cells with vacuoles. They recorded that embryonic and adult cells looked similar at the beginning of the culture, but would eventually differ from one another later, possibly confirming some of our results. Due to the far higher number of cases, our own experiments clearly showed the relationship of cell morphology to the donor’s age. The culture of human lens epithelial cells from cataract lenses, which were discussed in this paper, revealed a particularly large number of pathological cells. especially amongst older patients. In contrast to this, the stained flat preparations of lens capsules of cataract lenses (Konofsky. 1986) as well as normal eyes (Henke et al., 19 8 7) showed hardly any differences between the cell appearances of younger and older patients. We conclude that lens epithelial cells of cataract lenses probably have a higher sensitivity increasing with the age of the donor. They are only likely to take on pathological features when growing in these environmental conditions. It seems that tissue culture is able to represent exactly vitality and morphological alterations of the culture cells. Our observation of augmentation of cell diameter with increased age corresponds to the finding that cornea1 endothelial cells augment their diameter in increasing age. Acknowledgements We are very grateful to Mrs B. Gossler’s help in preparing the cultures, to Mr Kleider, Institut fur Medizinische Statistik. Universitat Erlangen-Niirnberg, for his great help in statistical evaluation and to Dr Adams for having corrected our poor English. References Bermbach, G. ( 1989). Humane und bovine Linsenepithelzellen in der Gewebekultur. Ph.D. Thesis.UniversitgtErlangenNiirnberg. Cogan. D. G.. Donaldson,D. D. and Reese,A. B. (1952). Clinical and pathological characteristicsof radiation cataract. Arch. Ophthalmol.47, 55-70. Eguchi.G. and Kodama,R. (1979). A study of human senile cataract: growth and differentiation of lens epithelial cellin in vitro cell culture. Ophthalmic Res. 11, 308-l 5. Fagerholm,P. P. and Philipson.B. T. (1981). Human lens epithelial in normal and cataractous lenses.Invest. Ophthabnol.
Vis. Sci. 21, 408.
Francois.J. and Victoria-Troncoso,V. (1979). Cytological study of senilecataract. Lloc. Ophthahnol. 47, 169-87. Francois.J., Victoria-Troncoso,V. and Cansu.K. (1978). Tissueculture of the epithehum of the normal and cataractousadult lens.Ophthahnologica 177. 2 14-22.
HUMAN
LENS
EPITHELIAL
CELLS
Franqois, J., Victoria-Troncoso, V., Cansu, K. and Lentini, F. (19 79). Morphological and microcinematographical tissue culture study of the differentiation of adult lens epithelial cells into fibres. Ophthalmic Res. 11, 341-50. Friedburg, D. and Mayer, U. (1971). Dei Wirkung von /3Naphthochinon auf glykolytische Enzyme des Linsenepithels Albrecht van Gruefes Archiv. KZin. Exp. OphthaZmol. 183, 152-7. Gospodarowicz, D., Mescher, A. L., Brown, K. D. and Birdwell C. R. (1977). The role of fibroblast growth factor and epidetmal growth factor in the proliferative response of the cornea1 and lens epithelium. Exp. Eye Res. 25, 63149. Hamada Y. and Okada, T. S. (19 78). In vitro differentiation of cells of the lens epithelium on human fetus. Exp. Eye Res. 26, 91-7. Henke, V. M., Engel, B., Guggenmoos-Holzmann. I. and Naumann. G. 0. H. (198 7). Quantitative cytology of flat preparations of the human lens capsule. Ber. anl. der AER. Koch, H.-R. (1979). Basic research on the crystalline lens and its importance for clinical ophthalmology. Ophthalmic Res. 11, 254-63. Konofsky, K. (1986). Quantitative Dichte-Bestimmung am Fluchpriiparut der menschlichen Kuturuktlinse. Ph.D. Thesis. Universitgt Erlangen-Niirnberg. Kuwabara, T. (1976). The maturation of the lens cell: a morphologic study. Exp. Eye Res. 20, 42743. Mayer, U. (1969). Untersuchungen zur Atmung an iiberlebendem Linsenepithel. Ber. Dtsch. Ophthul. Ges. Heidelberg 70, 359-61. Mayer, U. (1971). Messungen des CO,-Verbrauches von iiberlebendem Linsenepithel. Albrecht von Gruejes Arch. Klin. Exp. OphthuZmoZ. 183, 137-139. Mayer, U. (1974). Untersuchungen an Gewebekulturen UUS Kiilberuugen. Augural dissertation. Universittit Erlangen-Niirnberg. Mayer, U. and Sames, K. (1981). Sauerstoffverbrauch und Alterung in Langzeitkulturen von Linsenepithel. Albrecht von Gruefes Arch. KZin. Ophthulmol. 217, 117-24. Mungyer, G. and Jap, P. H. K. (1975). Morphologische und histochemische Aspekte des zellukiren Alterns in Kalbslinsenepithelkulturen. Verh. Anut. Ges. 69, 673-7. Naumann, G. 0. H. (1980). 9, Kapitel ‘Linse’. In Puthologie des Auges. Pp. 501-53. Springer-Verlag: Heidelberg, Berlin, New York. Papadonstantinou, J. (1967). Molecular aspects of lens cell differentiation. Science 156, 338-56. Paul, J. (1981). ZeZZ- und Gewebekultur. Verlag Walter de Gruyter : Berlin. Perry, M. M., Tassin, J. and Courtois, Y. (1979). A comparison of human lens epithelial cells in situ and in vitro in relation to aging : an ultrastructural study. Exp. Eye Res. 28, 32741. Platsch. U. D. and Wiederholt. M. (1981). Intracellular potassium altrory and cell membrane pot, of the isolated human rabbit lens. Exp. Eye Res. 33, 467-78. Ramaekers, F. C. S., Hukkelhoven, M. W. A. C., Groeneveld, A. and Bloemendal. H. (19 79). Cytoskeletal structures
119
in cultured bovine lens cells and their role in lens cell elongation. Ophthalmic Res. 11, 283-7. Reddan, J. R. and McGee, S. J. (1979). Human lens epithelial cells in tissue culture. J. CeZZBiol. 83, 112a. Reddan, J. R.. McGee, S. J., Goldenberg, E. M. and Dziedzic, D.C. (1982/83). Both human and newborn rabbit lens epithelial cells exhibit similar limited growth properties in tissue culture. Curr. Eye Res. 2, 399405. Rink, H. (1978). Lens epithelial cells in vitro. Znterdiscipl. Topics Gerontol. 12, 2438. Rohen, J. W. and Liitjen-Drecoll, E. (1982). Kup. Rinse in Funktionelle Histologic. Schatthauer Verlag : Stuttgart, New York. Ringens, P. J., Mungyer, G., Jap. P., Ramaekers, F., Hoenders, H. and Bloemendal, H. (1982). Human lens epithelium in tissue culture : biochemical and morphological aspects. Eqx Eye Res. 34, 201-7. Shapiro, A. L.. Siegel, I. M., ScharlT, M. D. and Robbins. E. (1969). Characteristics of cultures lens epithelium. Invest. OphthaZmoZ. 8, 393400. Stewart, S., Duncan, G., Marcantonio, J. M. and Prescott, A. R. (1988). Membrane and communication properties of tissue cultured human lens epithelial cells. Invest. Ophthulmol, Vis. Sci. 29, 1713-25. Streeten, B. W. and Eshagian, J. (1978). Human posterior subcapsular cataract: a gross and flat preparation study. Arch. Ophthulmol. 96, 1653-8. Tassin, J.. Malaise, E. and Courtois. Y. (1979). Human lens cells have an in vitro proliferative capacity inversely proportional to the donor age. Exp. Cell Res. 123, 388-92. van der Veen. J. and Heyen, C. F. A. (1959). Lens cells of the calf in continuous culture. Nature 183, 1137-8. van Venrooij. W. J., Groeneveld, A. and Bloemendal, H. (1974). Cultures calf lens epithelium. I. Methods and cultivation and characteristics of the cultures. Exp. Eye Res. 18, 517-26. van Venrooij, W. J.. Groeneveld, A. and Bloemendal, H. (1974). Cultured calf lens epithelium. II. The effect of dexamethasone. Exp. Eye Res. 18. 527-36. von Sallmann, L. (1952). Experimental studies of early lens changes after Roentgen-irradiation. III. Effect of Xradiation on mitotic activity and nuclear fragmentation of lens epithelium in normal and cystein-treated rabbits. Arch. OphthaZmoZ. 47, 305-20. von Sallmann, L. (1957). The lens epithelium in the pathogenesis of cataract. Am. 1. Ophthulmol. 44, 159. von Sallmann, L.. Halver, J. E., Collins, E. and Grimes, P. (1966). Thioacetamide-induced cataract with invasive proliferation of the lens epithelium in rainbow trout. Cancer Res. 26, 1819-5. von Sallmann, L., Tobias, C. A., Anger, H. 0.. Welch, C., Kimura, S. F.. Munoz, C. M. and Drungies. A. (195 5). Effects of high energy particles, X-rays and aging of lens epithelium. Arch. Ophthulmof. 54, 489-514. Vornhagen, R. and Rink, H. (1979). The influence of culture conditions on the behaviour of lens epithelial cells. Ophthalmic Res. 11, 288-92. Worgul, B. V. and Rothstein, H. (1975). Radiation cataract and mitosis. Ophthulmol. Res. 7, 21-32.
Human GEORG
Lens
BERMBACH,
Epithelial URSULA
University
Eye Clinic
(Received
1 March
MAYER
Erlangen1989
Cells
Niirnberg,
and accepted
AND
in Tissue
Culture
GOTTFRIED
0. H. NAUMANN
D-85200 in revised
Erlangen, form
Germany
72 April
1990)
In 94 cultures of human cataractouslensepitheiial cellsderived from patientsof different age,the cell diameterand morphology were observedand photographedafter 21 days. The following resultswere obtained: every culture had its typical pattern dependingon the ageof the patient. which was from 3 monthsto 94 yr. The cell culture taken from a young man was characterizedby smallerepitheloidcells showing a distinctly visible nucleus: the homogeneityof the monolayer diminishedin parallel to the increasing age of the patients. The cell morphology becameincreasingly inhomogenous,showed polymorphismand signsof vacuolic degeneration,and averagecell diameterincreasedin parallel with the increasingage.The cellsshowedthree characteristicphenotypes(Fig. 9), the morphologydepending significantly on the age of the culture and of the donor: small epithelial-likecellswithout vacuoles showing a distinctly visible nucleus (most of these had a diameter of SO-100pm), large cells with vacuoles,and cellswithout a visiblenucleusand in rare casesincluding vacuoles(they appearedround and irregular, their diameterwas 200-300 pm, and they included filamentousstructures). Longterm cultureswere maintainedfor 8 monthsand subculturesmaintainedup to the third passage. Key words:human lens: lensepithelialcells: lens: human lensepithelialcells: cataract; cell culture: tissueculture.
1. Introduction The lens derives from ectoderm and consists only of epithelial cells and their products. It is completely separated from all other types of cells by the basement membrane, which acts as a boundary layer. Therefore, lens epithelia can be cultivated without interruption of any nerval or vascular supply and without contamination from other types of cells like fibroblasts (Mayer, 1969 ; Reddan and McGee, 1979). For this reason, cells of such cultures are exclusively epithelial cells. Due to these anatomical facts, the lens is particularly suitable as a model for researching different scientific questions such as growth, mitosis and differentiation as well as protein synthesis and electrolyte transport (Mayer, 1969, 1974; Koch, 1979; Platsch und Wiederholt, 1981). Koch (19 79) described the importance of theoretical, as apposedto functional, basic research of the lens, especially concerning clinical ophthalmology. Cataracts are the most common diseaseof the lens and one of the most frequent causes of blindness (Naumann, 1980). The pathological mechanisms causing the most frequent types of cataract, especially the senile cataract, remains unknown. However, the function of lens epithelia is often discussedin connection with the pathogenesis of cataract (von Sallmann, 19 52, 19 5 7 ; Cogan, Donaldson and Reese, 1952; von Sallmann et al., 1955; Worgul and Rothstein, 19 75; Streeten and Eshagian, 19 78). The purpose of the present studies was to cultivate human lens epithelial cells obtained from surgically removed cataracts and to determine whether morpho00144835/91/020113+07
$03.00/O
logical changes in culture depend on the donors age and/or number of subcultures. The cell cultures system used was basedon that described by Mayer in 1969. This method has been used successfully to culture tissue removed from patients aged 3 months to 94 yr. The question was: do morphological alterations of the cells depend on the donor’s age or on the number of subcultures, and can parallels be found between both ? 2. Materials and Methods The cultures of human lens epithelial cells were all derived from cataractous eyes. The patients were operated on in the University Eye Clinic of ErlangenNiirnberg (Professor G. 0. H. Naumann), and the cataractous lens was removed by extracapsular extraction, i.e. the whole anterior capsule with the adhering subcapsular layer of epithelial cells was cut and removed from the lens and could be cultivated in one piece. The human lens capsules were set as explants in single cultures, with 94 samples, which grew out in culture, being examined. The graph shows the distribution of the age of those patients, whose cells grew out in vitro (Fig. 1). The precondition for the outgrowth of the cells was the attachment of the capsule on the flask bottom. The capsule was spread out with capsule forceps with the cell layer upwards on the surface of a plastic culture flask (Falcon cell culture flask, 50 ml volume, 2 5 cm2, Becton & Dickinson, Heidelberg). Medium was added without touching the capsule. In order to do this, the first flask was set on its end in the incubator 0 1991 AcademicPressLimited BER 52
G. BERMBACH
culture
ET AL.
flask
medium
FIG. 2. Plastic culture flask containing adhering lens epithelial cells.
the capsule with
Age (yr)
FIG. 1. Distribution
of the ages of donor patients.
so that the medium was located under the prepared capsule. In this way the tearing off of the capsule from the surface could be avoided. The rising water vapour protected the tissue from drying, forming a wet chamber. After 1 hr the flask was laid down, so.that the medium covered the epithelium, which was now firmly attached to the bottom (Fig. 2). TCM 199 [Hank’s (1 x ) by Biochrom KG, Berlin] was used as medium for all cultures, with 20% foetal calf serum and specific supplements being added: 2rnM L-glutamine, 100 I.U. penicillin ml-‘, 100 g streptomycin ml-l, 20 mM NaHCO,, 18 mM Hepesbuffer. For subcultivating, the confluent monolayer of the culture was incubated with trypsin and the dissociated cells were split 1: 2, as described by Paul (198 1). The growth of the cultures and the morphology of the cells were studied by an inverted microscope (Leitz) with a phase contrast and an incorporated camera. Kodak Ektachrome SO Asa, having a sensitivity of 18 DIN, was used: Twenty-one days after inoculation, the cells of each culture were photographed and compared by means of biomicroscopic photos. To identify special phenotypes and diameters of cells, the photos and a photographed scalewere projected on the samescreen (Fig. 3). Statistical Procedure To determine whether the diameters of human lens epithelial cells increases with donor age, the largest diameters of individual cultured cells were measured, using sample size of 30-50 cells. The mean and standard deviation for each patient were determined, and Spearman’s rank correlation-coefficient was calculated with respect to age. To ascertain whether pathological cellsappear more
FIG. 3. Projection of the biomicroscope photographed scale to the same screen.
photo
and a
often in cultures of material removed from older patients, the frequency of the three different phenotypes was determined. The number of different phenotypes of cells was set in relation to the patient’s age. Again, using the rank-correlation by Spearman. the 9 5 % confidence interval was calculated and a test for correlation was done. 3. Results Descriptionof Individual Cultures After a 21-day Incubation, Typical Aspects (a) Cellsof a 3-month-old baby [Fig. 4 (A)]. The cells shown in Fig. 1 describe a more rapid and abundant outgrowth than in cultures obtained from older patients. An almost homogenous cell picture could be seenwith the cellsbeing rather small and regular, and the nucleus being distinctly visible in nearly all cells. Their shape was epithelial or polygonal, and their size did not exceed 100 pm on average. The nucleus was relatively large in contrast to the smaller cytoplasm surrounding it, meaning that the nucleocytoplasmic ratio had shifted towards the nucleus. The cells were generally well demarcated from each other, so that we could comment on the membrane. The membrane did not look smooth, but slightly lobulated. At that time neither vacuoles nor granules had appeared in the plasma. (b) Cells of a 42-yr-old man [Fig. 4 (B)]. The elements were well demarcated from each other, and were bigger than those previously demonstrated, reaching a size of 150 pm, but were still uniform. The shape was epitheloid and regular, the nucleus was
HUMAN
LENS
EPITHELIAL
CELLS
FIG. 4. A, Cells of a 3-month-old
FIG. 5. A, Cells of a 62-yr-old
115
baby, x 400. B, Cells of a 42-yr-old
man, x 400. B, Cells of a 73-yr-old
man, x 400.
man, x 400.
mostly visible, and the homogenous cell picture still existed. There were only a few cells containing vacuoles.
cellswithout a distinctly visible nucleus, often showing filamentous structures. There were alsosomeepithelial cells, which were small and regular.
(c) Cells of a 62-yr-oZd mun [Fig. 5 (A)]. Now there was an heterogenous cell picture with pathological cells dominating: they contained vacuoles and were enlarged and irregularly shaped. The pathological cells had a diameter of 200-300 pm and were large
(d) Cells of a 73-yr-oZd man [Fig. 5 (B)]. In this culture, there was no uniform type of cell. The mixed population included apparently young appearing epithelial cells, although these were somewhat larger than their normal counterparts and possesseda large 8-2
116
G. BERMBACH
ET AL.
FIG. 6. A, Cellsof a 94-yr-old woman. x 400. B, Third subculture: 69-yr-old man, 8 months in culture, x 400.
nucleus. Enlarged cells containing cytoplasmatic vacuoles were also observed. In thesecells, the nucleus was rarely visible. The pathologic cells were large, heterogenous, irregularly shaped and were transparent with cytoplasm accumulating at the periphery. (5) Cellsof a 94-yr-oZd woman [Fig. 6 (A)]. Growth capacity was found even in the cells of this 94-yr-old woman. In the centre there was a typically vacuolated cell, surrounded by someenlarged senilecells, without a distinctly visible nucleus. Moreover, some smaller epithelial shaped cells were observed. As already shown in the cells of the above-mentioned patient, the cell picture showed inhomogeneity and could be classifiedin the old-age group. Gapswere found in the layer, which means a confluent monolayer had not been formed. Signs of vacuolic degeneration were predominant. Morphological Descriptionof the Passages Cellular growth extending from the explants occurred after 2-5 days, regardlessof the patient’s age. Initially they were similar to fibroblasts but later they lay together like epithelial cells. forming a monolayer. Mostly the cells formed a confluent layer after 3 weeks, also independent of the patient’s age. With increasing time in culture, signs of cell ageing increased much more rapidly in cells of older patients than in those of younger ones. After only 5 weeks many large and bulgy cellswith filamentous structures could be found in samples of older patients. After 5 months the above-mentioned large transparent cells with smooth membrane and filamentous structures were predominant in the cell picture. Where the
nucleus was still visible, it was surrounded by many vacuoles. In this obviously stationary phase the samplesremained in culture for 8 months. Third subculture [Fig. 6 (B)]. In the third subculture only large senile cells could be found, which were transparent and rigid. The proliferation capacity had apparently stopped and further subculture could not be established. The cells remained in this stationary phase for several months. The morphological appearance of the cell cultures depended on the age of the donor patient. Cultures of material obtained from patients of 3 months to 30 yr of age were homogenous, possesseda minimum number of pathological cells and had an average cell diameter not exceeding 100 pm. Cultures of material removed from patients between 30 and 55 yr of age were still homogenous. but possessedZO-50% pathological cells, and the average cell diameter in these cultures was greater, at 150 pm. Heterogenous cultures were observed when donor material from patients aged 50-65 yr was used: pathological cells dominated and the average cell diameter was 1SO-250 pm. Finally, donors beyond 65 yr of age yielded cultures consisting predominantly of pathological cells (90%). Cell diameters ranged between 100 and 400 ;ern and the cultures were extremely heterogenous. Statistical Evaluation First question: Doesthe cell diameterincreasewith the ageof the donor? We measured the longest diameter of the smallest and of the largest cell in every culture. Having done this, the mean cell diameter of 30-50
HUMAN
350
LENS
EPITHELIAL
CELLS
117
-
x x x x x
x
x $?ix
xdXx xx! ,“x I
30
40
50
/
Ld’” x-x 2~x.x ’ xx ;x x
x
x
x
xx I
I
I
I
60
70
80
90
I
Age group
Age (yr)
FIG. 7. Correlation of donor patient.
between average cell diameter and age
Spear-man’srank correlation coefjcient Spearman correlationscoefficient
Confidenceinterval (95%)
0.516 0.788
0.290-0611 0664-0837
-0.754
of phenotypes
in cor-
TABLE II
TABLE I
Range Mean cellaverage Phenotype 1 (relative frequency) Phenotype 2 (relative frequency) Phenotype 3 (relative frequency)
FIG. 9. Mean relative frequency relation to donor patient’s age.
- 0.604-(
Correlation coefficient, 95 % interval, and P-value of the different phenotypes Age group (Yd O-30
- 0.804) 31-55
0.411
0188-0.598
O-674
0~530-0-597
56-65 > 65
Phenotype 1 2 3 1 2 3 1 2 3 1 2 3
n
6 15 16 57
Mean relative frequency
S.D.
0.873 0.111 0.016 0.656 0.185 0.159 0.434 0.189 0.377 0.166 0.2 70 0.564
0.101 0.101 0028 0.097 0.08 5 0.062 0.158 0090 0.186 0.163 0.137 0.177
P < 0.01.
CELLS WITH DISTINCTLY NUCLEUS; SMALLER; EPITHELIAL MORPHOLOGY
CELLS
WITH
CELLS OFTEN
WITHOUT VISIBLE WITH FILAMENTDUS
FIG.
Secondquestion: Dopathologicalcellsappearmoreoften in cultures taken from older or younger patients? We counted the number of the phenotypes 1, 2 and 3 in every culture (Fig. 8). In each age group, the mean relative frequency (Fig. 9) and the corresponding standard deviation was calculated. The number of phenotype 1 decreasedwith the donor’s age, whereas frequence of phenotypes 2 and 3 increased. In order to prove this, the correlations-coefficient (rank-correlation by Spearman), the 9 5 % confidence-interval and the P-value were calculated. Significant differences in the number of different phenotypes of cells depending on the age of the donor patient were found (Table II).
VISIBLE
VACUOLES
8. Characteristic
NUCLEUS; STRUCTURES
phenotypes
of cells.
4. Discussion cells and the range, that means the variation of the cell diameter in this sample, were calculated. We found that the mean cell diameter did increase with the age of the patient (Fig. 7). Spearman’s rank correlationcoefficient was calculated (Table I) which confirmed that there was a significant (P < 0.01) correlation between the average cell diameter and the age of the donor patient.
The cell culture technique was tist described in 1969 by Mayer who examined the oxygen uptake of cultured bovine lens epithelia. By Warburg’s respirometer she determined the respiratory quotient of the lens epithelium to be of 1.04 (1971). By taking photographs every 30 min for 2 days, Mayer (19 74) found a middle division range of cell division of 10.5 hr. Mayer and Sames (198 1) examined oxygen
G. BERMBACH
118
consumption and ageing in bovine lens epithelia and observed that the oxygen consummation was without any alteration up to the 40th passage. These were our parameters of cell growth. All photos were taken after a 21-day culture, and growth conditions were the same in all cases. It is difficult to perform comparative studies using relatively normal tissue. Clear lens tissue is never surgically removed and donor eyes require extended transport-times and hence the tissue is difficult to culture successfully. The modified method for cultivating human lens epithelial cells described in this paper makes an application for tissue culture possible. which is practicable in a large number and reproduces well. Very little comparable literature can be found on tissue culture of human lens epithelial cells as observed by Ringens et al. (1982). He cites Hamada and Okada (1978). Tassin, Malaise and Courtois (19 79). Eguchi and Kodama (1979) and Perry, Tassin and Courtois (19 79). FranGois and Victoria-Troncoso ( 19 79) are, however, not cited. Only Reddan et al. (1982/83) can be found in recent literature. However, all these authors obtained the operated cataract lenses by cryoextraction. Using this method always destroys a part of the epithelia. In extracapsular cataract extraction the total microsurgical removal of the lens capsule with the adhering subcapsular layer of epithelia is a much more adequate technique. The often used enzymatic dissociation of the epithelial cells from the capsule by trypsine (Hamada and Okada. 1978; Eguchi and Kodama, 1979) damages the cells (Paul, 198 1) and was not successful in our own experiments. The cells need time to recover, but they can also be permanently impaired. Franqois et al. (1979) described a very costly method, which involved sticking the capsule on the flask bottom, but this did not prove successful in our own experiments. Reddan et al. (1982/83) examined the morphology of the cells in culture in eight different media with various content and growth factors: according to his study, human lens cells obtained from normal and cataract lenses showed a similar appearance. However, they had a limited growth capacity in vitro, in spite of the enrichment of the medium by well-known mitogens. Therefore, Reddan came to the conclusion that the present media were still lacking in some essential substrates for the long-term culture of human lens cells. We conclude that lens cells cannot be cultivated as abundantly as tumour cells could be. Such cultures could help in the understanding of factors controlling growth, development, repair and ageing of these cells, and would also make it possible to prove genetic defects connected with congenital cataracts. Ringens et al. (1982) cultivated human foetal lens cells, which also showed limited growth capacity in vitro. He described signs of cell ageing which appeared after 3 months in the primary culture as well as in the
ET AL.
subculture, and also observed vacuolic degeneration and filamentous structures, similar to the observations described above. He did not measure any cell diameter : neither did Hamada and Okada (19 78), Eguchi and Kodama (1979) and Paul (1981). Perry et al. ( 19 79 succeeded in incubating foetal and adult human lens cells, and observed a heterogenous cell picture after 10 weeks, the cultures consisting of young appearing cells, and cells with vacuoles. They recorded that embryonic and adult cells looked similar at the beginning of the culture, but would eventually differ from one another later, possibly confirming some of our results. Due to the far higher number of cases, our own experiments clearly showed the relationship of cell morphology to the donor’s age. The culture of human lens epithelial cells from cataract lenses, which were discussed in this paper, revealed a particularly large number of pathological cells. especially amongst older patients. In contrast to this, the stained flat preparations of lens capsules of cataract lenses (Konofsky. 1986) as well as normal eyes (Henke et al., 19 8 7) showed hardly any differences between the cell appearances of younger and older patients. We conclude that lens epithelial cells of cataract lenses probably have a higher sensitivity increasing with the age of the donor. They are only likely to take on pathological features when growing in these environmental conditions. It seems that tissue culture is able to represent exactly vitality and morphological alterations of the culture cells. Our observation of augmentation of cell diameter with increased age corresponds to the finding that cornea1 endothelial cells augment their diameter in increasing age. Acknowledgements We are very grateful to Mrs B. Gossler’s help in preparing the cultures, to Mr Kleider, Institut fur Medizinische Statistik. Universitat Erlangen-Niirnberg, for his great help in statistical evaluation and to Dr Adams for having corrected our poor English. References Bermbach, G. ( 1989). Humane und bovine Linsenepithelzellen in der Gewebekultur. Ph.D. Thesis.UniversitgtErlangenNiirnberg. Cogan. D. G.. Donaldson,D. D. and Reese,A. B. (1952). Clinical and pathological characteristicsof radiation cataract. Arch. Ophthalmol.47, 55-70. Eguchi.G. and Kodama,R. (1979). A study of human senile cataract: growth and differentiation of lens epithelial cellin in vitro cell culture. Ophthalmic Res. 11, 308-l 5. Fagerholm,P. P. and Philipson.B. T. (1981). Human lens epithelial in normal and cataractous lenses.Invest. Ophthabnol.
Vis. Sci. 21, 408.
Francois.J. and Victoria-Troncoso,V. (1979). Cytological study of senilecataract. Lloc. Ophthahnol. 47, 169-87. Francois.J., Victoria-Troncoso,V. and Cansu.K. (1978). Tissueculture of the epithehum of the normal and cataractousadult lens.Ophthahnologica 177. 2 14-22.
HUMAN
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EPITHELIAL
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Franqois, J., Victoria-Troncoso, V., Cansu, K. and Lentini, F. (19 79). Morphological and microcinematographical tissue culture study of the differentiation of adult lens epithelial cells into fibres. Ophthalmic Res. 11, 341-50. Friedburg, D. and Mayer, U. (1971). Dei Wirkung von /3Naphthochinon auf glykolytische Enzyme des Linsenepithels Albrecht van Gruefes Archiv. KZin. Exp. OphthaZmol. 183, 152-7. Gospodarowicz, D., Mescher, A. L., Brown, K. D. and Birdwell C. R. (1977). The role of fibroblast growth factor and epidetmal growth factor in the proliferative response of the cornea1 and lens epithelium. Exp. Eye Res. 25, 63149. Hamada Y. and Okada, T. S. (19 78). In vitro differentiation of cells of the lens epithelium on human fetus. Exp. Eye Res. 26, 91-7. Henke, V. M., Engel, B., Guggenmoos-Holzmann. I. and Naumann. G. 0. H. (198 7). Quantitative cytology of flat preparations of the human lens capsule. Ber. anl. der AER. Koch, H.-R. (1979). Basic research on the crystalline lens and its importance for clinical ophthalmology. Ophthalmic Res. 11, 254-63. Konofsky, K. (1986). Quantitative Dichte-Bestimmung am Fluchpriiparut der menschlichen Kuturuktlinse. Ph.D. Thesis. Universitgt Erlangen-Niirnberg. Kuwabara, T. (1976). The maturation of the lens cell: a morphologic study. Exp. Eye Res. 20, 42743. Mayer, U. (1969). Untersuchungen zur Atmung an iiberlebendem Linsenepithel. Ber. Dtsch. Ophthul. Ges. Heidelberg 70, 359-61. Mayer, U. (1971). Messungen des CO,-Verbrauches von iiberlebendem Linsenepithel. Albrecht von Gruejes Arch. Klin. Exp. OphthuZmoZ. 183, 137-139. Mayer, U. (1974). Untersuchungen an Gewebekulturen UUS Kiilberuugen. Augural dissertation. Universittit Erlangen-Niirnberg. Mayer, U. and Sames, K. (1981). Sauerstoffverbrauch und Alterung in Langzeitkulturen von Linsenepithel. Albrecht von Gruefes Arch. KZin. Ophthulmol. 217, 117-24. Mungyer, G. and Jap, P. H. K. (1975). Morphologische und histochemische Aspekte des zellukiren Alterns in Kalbslinsenepithelkulturen. Verh. Anut. Ges. 69, 673-7. Naumann, G. 0. H. (1980). 9, Kapitel ‘Linse’. In Puthologie des Auges. Pp. 501-53. Springer-Verlag: Heidelberg, Berlin, New York. Papadonstantinou, J. (1967). Molecular aspects of lens cell differentiation. Science 156, 338-56. Paul, J. (1981). ZeZZ- und Gewebekultur. Verlag Walter de Gruyter : Berlin. Perry, M. M., Tassin, J. and Courtois, Y. (1979). A comparison of human lens epithelial cells in situ and in vitro in relation to aging : an ultrastructural study. Exp. Eye Res. 28, 32741. Platsch. U. D. and Wiederholt. M. (1981). Intracellular potassium altrory and cell membrane pot, of the isolated human rabbit lens. Exp. Eye Res. 33, 467-78. Ramaekers, F. C. S., Hukkelhoven, M. W. A. C., Groeneveld, A. and Bloemendal. H. (19 79). Cytoskeletal structures
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in cultured bovine lens cells and their role in lens cell elongation. Ophthalmic Res. 11, 283-7. Reddan, J. R. and McGee, S. J. (1979). Human lens epithelial cells in tissue culture. J. CeZZBiol. 83, 112a. Reddan, J. R.. McGee, S. J., Goldenberg, E. M. and Dziedzic, D.C. (1982/83). Both human and newborn rabbit lens epithelial cells exhibit similar limited growth properties in tissue culture. Curr. Eye Res. 2, 399405. Rink, H. (1978). Lens epithelial cells in vitro. Znterdiscipl. Topics Gerontol. 12, 2438. Rohen, J. W. and Liitjen-Drecoll, E. (1982). Kup. Rinse in Funktionelle Histologic. Schatthauer Verlag : Stuttgart, New York. Ringens, P. J., Mungyer, G., Jap. P., Ramaekers, F., Hoenders, H. and Bloemendal, H. (1982). Human lens epithelium in tissue culture : biochemical and morphological aspects. Eqx Eye Res. 34, 201-7. Shapiro, A. L.. Siegel, I. M., ScharlT, M. D. and Robbins. E. (1969). Characteristics of cultures lens epithelium. Invest. OphthaZmoZ. 8, 393400. Stewart, S., Duncan, G., Marcantonio, J. M. and Prescott, A. R. (1988). Membrane and communication properties of tissue cultured human lens epithelial cells. Invest. Ophthulmol, Vis. Sci. 29, 1713-25. Streeten, B. W. and Eshagian, J. (1978). Human posterior subcapsular cataract: a gross and flat preparation study. Arch. Ophthulmol. 96, 1653-8. Tassin, J.. Malaise, E. and Courtois. Y. (1979). Human lens cells have an in vitro proliferative capacity inversely proportional to the donor age. Exp. Cell Res. 123, 388-92. van der Veen. J. and Heyen, C. F. A. (1959). Lens cells of the calf in continuous culture. Nature 183, 1137-8. van Venrooij. W. J., Groeneveld, A. and Bloemendal, H. (1974). Cultures calf lens epithelium. I. Methods and cultivation and characteristics of the cultures. Exp. Eye Res. 18, 517-26. van Venrooij, W. J.. Groeneveld, A. and Bloemendal, H. (1974). Cultured calf lens epithelium. II. The effect of dexamethasone. Exp. Eye Res. 18. 527-36. von Sallmann, L. (1952). Experimental studies of early lens changes after Roentgen-irradiation. III. Effect of Xradiation on mitotic activity and nuclear fragmentation of lens epithelium in normal and cystein-treated rabbits. Arch. OphthaZmoZ. 47, 305-20. von Sallmann, L. (1957). The lens epithelium in the pathogenesis of cataract. Am. 1. Ophthulmol. 44, 159. von Sallmann, L.. Halver, J. E., Collins, E. and Grimes, P. (1966). Thioacetamide-induced cataract with invasive proliferation of the lens epithelium in rainbow trout. Cancer Res. 26, 1819-5. von Sallmann, L., Tobias, C. A., Anger, H. 0.. Welch, C., Kimura, S. F.. Munoz, C. M. and Drungies. A. (195 5). Effects of high energy particles, X-rays and aging of lens epithelium. Arch. Ophthulmof. 54, 489-514. Vornhagen, R. and Rink, H. (1979). The influence of culture conditions on the behaviour of lens epithelial cells. Ophthalmic Res. 11, 288-92. Worgul, B. V. and Rothstein, H. (1975). Radiation cataract and mitosis. Ophthulmol. Res. 7, 21-32.
