An Established Cell Line from the Newt Notophthalmus vin'descens D. H. REESE* Laboratory of Vision Research, National Eye Institute, National Institutes of Health, Department of Health, Education and Welfare, Bethesda, Maryland 20014, USA

T. YAMADA and R. MORET Institut Suisse de Recherches Experimentales sur le Cancer, CH 1011 Lausanne, Switzerland

Received February 1976lRevised March 1976

An established cell line, derived from the dorsal iris of the eastern North American newt, Notophthal- mus viridescens, is described. The cells display an epithelial-like behaviour in culture, grow relatively slowly, possess considerably larger chromosomes than mammals and are heteroploid, although some near-diploid cells are present in the culture. The line is characterized by a strong tendency to overlap- ping and aggregation in spite of its origin from adult tissue.

Introduction

Some species of amphibians belonging to the order Uro- dela possess the largest of all vertebrate somatic cells, and the highest amount of genome DNA of all eukary- otes. With these properties, the somatic cells of uro- deles have high potential as material for cell biological investigation. For example, it has been found recently that newt somatic cells in culture are exceptionally well suited for the micro-injection method established by Grassmann and Grassman, for mammalian cells [ l l . Such advantages could be more effectively utilized if established cell lines derived from somatic tissues of uro- deles were available. While there are numerous amphib- ian cell lines now in existence [2, 31, all have been de- derived from anuran tissues, which do not have the above properties. This paper describes a line from the adult eastern North American newt, Notophthalmus virides- cens. An unusual feature of the cell line is that it is prob- ably derived from iris epithelial cells which have been shown to undergo complete dedifferentiation and to be- come lens cells during lens regeneration [4, 5 , 6 , 71. The line is thus valuable for the analysis of the dedifferentia- tion pocess.

Methods

Source of Tissue. Adult eastern North American newts were collect- ed in eastern Tennessee and maintained in the laboratory as de- scribed previously [81.

* Present address: National Cancer Institute, National Institutes of Health, Bldg. 37, Room 3C02, Bethesda, Maryland 20014, USA

Preparation and Maintenance of Cultures. The entire dorsal halves of 29 irises were removed and cleaned according to the procedure previously described 191. The iris tissue contained, in addition to the pigmented iris epithelium and stroma, a small portion of the pars ciliaris. The tissues were dissociated by constant stirring with the aid of a magnetic stirring bar at 25O C in 5 ml of 0.25% trypsin solution made up in half-strength, calcium- and magnesium-free (Ca-Mg- free), phosphate-buffered saline (PBS). At 15-min intervals, the dis- sociation medium, containing free cells, was decanted and centri- fuged at 300 g. Cells were resuspended in 4 ml of amphibian-ad- justed Leibovitz L-15 culture medium [lo] containing 10% foetal calf serum and 1 96 Gibco IOOX Antibiotic-Antimycotic solution. Cultures were maintained at 23-24O C in 30-ml plastic flasks with loosened caps to allow the gas phase inside the flasks to equilibrate with ambient air. When cultures approached confluence, cells were subcultured by dissociation in 0.05% trypsin made up in Ca-Mg-free PBS and resuspended in culture medium at dilutions of 1 : 5 or 1 : 10. All tissue-culture reagents were obtained from Grand Island Biological Co. Tissue culture dishes and flasks were obtained from Falcon Plastics, Inc.

Melhods for Scanning Electron Microscopy. Cells were placed on to cover slips in 35 mm plastic dishes and cultured as indicated, then fixed with a mixture of 2% OsO, and 2% HgCl, for 1 h at 4 O C . They were then rinsed with 0.05 M phosphate buffer at pH 7.4, dehydrated through an ethanol series, treated with 40-10096 amyl acetate in absolute ethanol, dehydrated in liquid C 0 2 with the critical point method, fixed on the scanning support and coated with gold. The model JS M-35 was used for observation.

Chromosome Preparation. Cells growing in a 250-ml flask in log phase and at near confluence were treated with colcemide (0.6 pdml of culture medium) for 6 h. After dissociation with trypsin and two washings with Ca-Mg-free PBS, the cells were resuspended in 10 ml of 0.27% KCI and allowed to swell at room temperature for 30 min.

Differentiation 6, 75-81 (1976) - 0 by Springer-Verlag 1976

76 D. H. Keese et al.:

Then 0.2 ml of fixative (3 methanol : 1 acetic acid) was added to the KCI solution and, after centrifugation, the cells in the pellet were resuspended in 5 ml of furative and stored at 4’ C overnight. After two changes of fresh fixative, the cells were resuspended in 5 ml of fresh fixative, and spreads of metaphase chromosomes were pre- pared by the ignition-air dry method [ 111. After staining with Giem- sa stain. metaphase figures were counted at random to obtain an estimate of the range of chromosome number in the cell popula- tion.

Results

Cells were first put into culture on September 14, 1967. A total of four 15-min harvests was required for com- plete dissociation of the iris tisue. While any one of the four harvests contained enough cells to start cultures, the second produced a culture in which epithelial-like cells containing melanosomes were in the majority. The first harvesting yielded mostly erythrocytes and nonpig- mented fibroblast-like cells. The cell line in existence now is from the second harvesting and is designated TVI. Cells attached within several hours. A period of approximately two weeks then ensued, during which pigmented cells were seen to contain, with time, fewer melanosomes. During this same period, large numbers of free melanosomes were seen in the medium. After approximately two weeks, the cells appeared to be com- pletely free of melanosomes. No identifiable melano- cytes have reappeared in the culture. Although no

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Fig. 1. Abscissa: days in culture; ordinate: cells per dish x lo-’. Growth curve for TVI line at passage 59. Cells were seeded into 60 mm dibhes at a density of 3.3 x lo5 cells/dish. Each point represents the mean cell count of 3 dishes. Vertical bars show range of measure- ments

Fig. 2. Confluent culture showing piling-up of cells into spherical bodies. Arrows: note rounded-up cells in mitosis. Phase contrast. Line is ca. 40 pm

Established Newt-Cell Line

Fig. 3. Edge of a monolayer showing epithelial-like behaviour of TVI cells. Phase contrast. Line is ca. 40 pm

Fig. 4. Near-diploid cell in mitosis. Methanol-acetic acid (3 : 1). Hematoxylin eosin. Line is ca. 10 pm

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Figs. 5-8. Legends see page 79

D. H. Reese et al.:

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Established Newt-Cell Line

Table 1. Chromosome frequency distribution

Number of cells: 2 1 1 1 1 1 2 2 1 1 2 4 1 1 3 6

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Numberofchromosomes: 10 15 16 20 21 23 24 29 30 37 38 40 43 45 uncountable

growth measurements were made, it was possible during the early months of culture to subculture cells at dilu- tions of as high as 1 : 20 and achieve confluence after three to four weeks. This period of relatively active growth lasted for approximately six months, then growth decreased and has remaind constant. Figure 1 shows the growth curve for cells at the 59th passage. The population doubling time for the cells in log phase (calculated from Fig. 1) was 84 h. There was a marked tendency for growth to continue after cells became con- fluent; the result was a considerable piling up and aggre- gation of cells into spherical bodies (Fig. 2). This behav- iour still persists. Under monolayer conditions of growth, the cells were epithelial-like (Fig. 3). Cells in the process of mitosis were either rounded-up (Fig. 2) or flattened (Fig. 4). Nuclear' diameter under monolayer conditions of growth was in the range of 20 x 40 Pm.

In the scanning electron micrographs, extensive vari- ation in the architecture of the cell surface of TVI cells is apparent. Irregular knobs (Fig. 5) , fine microvilli (Fig. 6), spherical blebs (Fig. 7, 8), etc., can be recognized. Combined phase contrast and scanning electron micro- scopic observations suggest a dynamic condition of the cell surface associated with extensive discharge of cell surface material.

In the preparation of metaphase chromosomes, air drying without ignition was totally unsatisfactory and while the ignition-air dry method greatly facilitated chromosome spreading, it was still difficult to obtain chromosomes that were sufficiently separated for count- ing. In most cases, metaphase chromosomes were entan- gled and clumped together. Of the 57 metaphase figures examined at random, more than half were uncountable, though obviously polyploid (Table 1). Those that were countable varied considerably in number, ranging from

near-haploid to near-tetraploid. There were, however, within the population, cells that were near-diploid. Fig- ure 9 depicts near-diploid metaphase chromosomes and, for the purpose of size comparison, chromosomes from a cultured rat liver cell.

Discussion

Although the predominant cell type in vitro during the initial period of culture was a melanocyte, because clon- ing was not performed on melanocytes during this peri- od and because these cells subsequently lost all melano- somes, it cannot be stated with certainty that the TVI cell line originated from iris epithelial cells. However, if one compares TVI cells with the depigmented primary cul- tures derived from iris epithelium separated from the stroma prior to enzymatic dissociation [12, 131, there exist close similarities in their structure as revealed by scanning electron microscopy (Yamada and Moret. un- published). Moreover, the movements of these two cells as observed by phase microscopy are indistinguishable. Hence it appears probable that TVI cells are derived from iris epithelial cells. The observed gradual disap- pearance of melanosomes from cells and the appearance of free melanosomes in the medium during the first weeks of culture suggests, but does not rigorously prove, that melanosomes were expelled from living pigmented iris cells in vitro. This possible mechanism of melano- some loss is emphasized because it has been well docu- mented that depigmentation of iris epithelial cells in vivo during Wolffian lens regeneration is mainly achieved by discharge of melanosomes 114, 151. It has been recog- nized, however, that dilution by cell replication also con- tributes to depigmentation in vivo [161. Both of these mechanisms may have played a role in the depigmenta-

Figs. 5-8. Scanning electron microscope view of TVI cells

Fig. 5. A part of a laminar cell with some knobs and marginal microvilli. The cell with spherical blebs left above is presumably in anaphase of mitosis, ca. 5400 x

Fig. 6. A part of the upper surface of a laminar cell showing a population of microvilli, ca. 18000 x

Fig. 7. A spherical cell probably in prophase with microvilli and irregular blebs, ca. 3600 x

Fig. 8. A small aggregate of cells formed on top of a sparce monolayer oflaminar cells. Some cells with spherical knobs are presumably in anaphase suggesting that cells do divide after aggregation, ca. 720 x

80 D. H. Reese et al.:

We attribute the different behaviours to loss of capacity of melanosome synthesis during prolonged cultivation in the case of TVI line. In the initial period of the cell line, cells were subcultured before they were completely con- fluent. This is possibly the reason that no repigmenta- tion was observed in that period of the TVI line.

The strong tendency for overlapping and aggrega- tion of TVI cells upon confluence indicates a complete lack of contact inhibition, even though these cells were derived from adult tissue. It is possible that the loss of contact inhibition is associated with heteroploidy. How- ever, it should be pointed out that in primary cultures of dorsal iris or in pure primary cultures of iris epithelial cells of adult newts, where heteroploidy should be at a minimum, cells aggregate upon confluence 17, 13, 171. One possible interpretation of the lack of contact inhibi- tion in all cultures from adult iris cells including the TVI line is related to depigmentation: in lens regeneration, iris epithelial cells give rise to the cells of lens tissue after depigmentation, and the lens tissue has the properties of an embryonic lens rudiment when formed [lSI. Thus, one could argue that depigmentation of iris epithelial cells is associated with rejuvenescence of these cells. If this is the case, contact inhibition would cease to operate in the depigmented progenies of iris epithelial cells. Al- though the aggregates of TVI cells appear similar to “the lens-like structures” obtained by Eguchi et al. [71, they do not show signs of lens differentiation in immu- nofluorescence tests (McDevitt and Reese, unpublished) and in ultrastructure (Dumont, unpublished).

Compared to most mammalian and some amphibian cell lines [3, 191, the growth rate of the TVI cell line is slow. The ambient temperature at which these cells are grown may, in part, explain the slow growth. However, some amphibian cells, for example the A6 and AS lines from Xenopus [31, are also grown at a low ambient tem- perature and have growth rates comparable to the most rapidly dividing mammalian lines. The slow growth rate may be more a function of the large amount of DNA in the newt cell; N . viridescens contains 98 picograms of DNA per diploid cell, 10 to 20 times the DNA content of most mammalian and Xenopus cells and approxi- mately 40 times that of chick cells [201. This high DNA content, coupled with the polyploid nature of the TVI cell, probably places severe demands on the processes required for cell replication. It is interesting that growth was more rapid during the early period of culture when there was a greater probability that the cells were dip- loid. Even under conditions of rapid growth in vivo the newt cell cycle is relatively long. During the most active phase of cell replication during Wolffian lens regenera-

Fig. 9. Metaphase chromosomes of two near-diploid TVI cells. Inset shows metaphase chromosomes of a rat liver cell. Line is ca. 10 pM

tion of TVI cells. Although confluence is not accompan- ied by the repigmentation of TVI cells, it has been repea- tedly observed that in primary cultures of dorsal iris cells [ 171 and in pure primary cultures of iris epithelial cells (Yamada, unpublished), repigmentation occurs after depigmented cells become confluent and aggregate.

Established Newt-Cell Line

tion, cell generation times range between 23 h and 79 h 116, 211. For the primary culture of newt iris cells, the cell generation time is estimated to be 69 h [171.

The combination of a high DNA content and a 2 N chromosome number of 22 [22] obviously accounts for the large size of newt metaphase chromosomes. This large size resulted in entanglement and clumping of chromosomes, making it difficult to obtain well-spread preparations consistently, at least with the techniques employed in this study. A systematic study of the condi- tions best suited for the preparation of newt chromo- somes should help to solve this problem. Because of the high percentage of uncountable metaphase figures, it is difficult to assess the degree of chromosomal instability. It is evident, however, that the line should be classified as heteroploid.

The TVT line has not been continuously grown in culture since it was first set up in 1967. During part of the time cells have been stored frozen in liquid nitrogen. The highest passage number achieved was 73. This cul- ture was subsequently lost at this passage due to fungal contamination. Fortunately, at various passages (1 7th, 26th, 29th), cells were frozen and stored for such situa- tions. The cells that are currently being carried are at a passage number greater than 50 and show no apparent decline in vigour from that reported in this paper.

In a recently completed work (Reese, unpublished), the RNAs of TVl cells have been extensively studied using isotopic labelling and acrylamide gel fractionation. The presence of labelled 23 S and 16 S ribosomal RNA, which is characteristic of prokaryotes, has never been detected in RNA extracted from these cells. Since this isotopic labelling procedure has been shown to be a very sensitive assay for the presence of mycoplasma [231, the absence of 23 S and 16 S ribosomal RNA from the TVI cells indicates that these cells are free of mycoplasma as well as other prokaryotic contamination.

Acknowledgements: This work was partly carried out at the Bio- logy Division, Oak Ridge National Laboratory, Oak Ridge, Tennes- see, USA and sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation, and partly at the Institut Suisse de Recherches Experimentales sur le Cancer was

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supported by Fonds National Suisse de la Recherche Scientifique (No. 3.0860.73).

Thanks are due to Dr. Alfred J. Coulombre for his support and encouragement, Dr. James Regan and Mr. William Lee for their help in carrying the TVI line, Drs. Haydon Coon and Keen Rafferty for helpful suggestions, and Mr. Jerry Elliott for the slide of rat cell chromosomes.

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