Department of Fisheries and Oceans, Central and Arctic Region, Freshwafer Institute, Winnipeg, Man. R3T 2N6

S. Fujioka an Water Resources Research Center, University of Hawaii, HonoEulu, Hd 96822 USA

Nielsen, O., 8. K. Kelly, W. 8. Lillie, ). W. Clayton, R. S. Fujioka, and . S. Yoneyama. 1989. Some properties of a finite cell line from beluga whale (Desphinapterus %pusas). Can. 1. Fish. Aquat. Sci. 46: 7 472-1 477

#) was derived from kidney of an immature male k I whale (Deiphinapterus hcas) . 19k 7 passages at 37°C in enriched tissue culture a supplemented with 10% fetal

bovine serum and containing antibiotics. The cell line had a diploid chromosome modal number (2n =44), but a pseudodiploid karyotype. BWK cells were characterized by lactate dehydrogenase (LBH), malate dehydrsgen- ase (MDH), glucssephosphate isomerase (GPI), and phosphsg[ucsmutaw (PGM) lsomyme systems. The cells were susceptible to infection by several rnarnrnalian viruses including coxsackievirus (B3 and B5), vaccinia, and herpes simplex 1 ; but were refractory to echovirus 27, influenza A, poliovirus (types 1 and 3), and simian agent 4 1. The BWK line, which has been stored in liquid nitrogen, appears to be suitable for isolation of viruses from marine mammals.

Une Zignee cellulaire f ink (BWK) a 6t6 tisee du rein d'un beluga m2Ie immature (DeBphapterus %eucas). Les cel lules ont kt6 cultiv6es par 4 9 k 4 passages 2 37°C dans un milieu de culture de tissus enrichi avec 18% de serum de foetus de bovin et contenant des antibiotiques. La lignee cellulaire pos chromosomes de type diplo'ide (2n = 44) mais un caryotype pseudodiploi'de. Les ce par des syst6mes d'isonyrnes de la lactate-deshydrogenase (L H), de la malate-dehydrog6 glucose phosphate issm6sase (GPI) et de la phosphogBucomutase (PCM Les cellules 6taien fecticsn par plusie~rrs virus mamrnaliens, Fsotamment Be virus Coxsackie ( 1 , mais etaient &fracctaires 3 I'6chovirus 27, au virus A de la grippe, au poliovirus (types 1 et 3) et 21 ['agent simien 11. La lignee BWK, qui est gad& dans l'amote liquide, sernble convenir 3 l'isolernent des virus chez les mam- rnif6res marins.

eeeived November 8, 1988 Accepted Apri% 26, 1 989 422933)

uable for research progress in

m the Atlantic spotted d Knimeff (1968) and

several marine cetacean species including the fin whale, Knriueff a d @my la mmwcrc~rhynehus,

delphis) and bottle siop gillk (now T. &runcatus) (Pirtle and Kniazeff 1968); gray whale, Eschrichtieas robustus (Amason 1

don msnoceros (Andrews et al. 1973); pea mysticeteas (Smith et A. 1987); Pacific

'A portion sf these results was presented at. the 37th Annual Meeting of the Tissue Culture Association, Chicago, IE., June 48, 1986.

hin, hgenorhyn us obliquidens (Cecil and uga whale, Delphinapterus ieuews) J x -

ell lines were specifically derived for short-term kqologicd or virological studies and were prop- agated only briefly in the labmtoq. ture applications have obviously been lack of availability of chxackrized

d noncontinuous). In the present study we describe , isszyme expression, cellular growth character-

istics, md viral susceptibility of a nite cell line that was derived from kidney tissue of beluga wh e (Nielsem et al. 1986). The present report establishes criteria for standardization sf eeta- cem cell lines and further dem~~nstraks the feasibility s f using such cqopresemed cetacean cells from early passage level cul- tures as defined systems for subsequent diagnostic virology and comparative biwhemPPist'$r.

edia

A previous report from the ATCC s that enriched tissue cultwe media was required for growth of the

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dolphin kidney (Sp l K) cell line (May et al. 1983). Therefore, we elected to use either of the following enriched, modified tissue culture media for culturing beluga whale kidney (BWK) cells:

(A) Growth medium I (GMl) consisted of Eagle's basal medium (EBM) with Esle's salts, enriched with dsuble- strength nutrients (i.e. m h o acids, 2 x ; vitamins, 2 x ; L-glutmine, 2 X ) and supplemented with 10% fetd bovine serum (EBS). (All ingredients were from GIBCO Laboratories, Life Tech., Inc., Grmd Island, NY.) (B) Growth medium 2 (GM2) consisted of Eagle's modified minimum essential medium (MEM), with Hanks' bdanced salt solution which was supplemented (per 1100 mL) with 2 mL of 200 &I L-glutamine (GIBCO); 10 m L of FBS; 2 mL of (100 x 1 amino acids for EBM (How Laboratories, McLem, VA); and 2 d of ( I 0 0 x ) vitamins for EBM (Flow Labs).

Each medium was buffered with sterile NdCO, solution md adjusted to pH 7 . S T . : ! prior to use. Cells were propagated in either of the above media (i.e. GMl or GM2) with added antibiotics, as subsequently specified.

Cell Culture Initiation a d Maintenance

W m q cell cultures were initiated from a kidney of an immature ((3.30 m, 54Q kg) male beluga whale (Nielsen et al. 1986). Cells were enzymatically dissociated from the minced kidney essue by modification of the method of Montes de Oca (1973) as previously described (Nielsen et d. 1986), The tissue was diced into small (2-3 mm3) pieces, rinsed several times in phosphate buffered saline. (PBS) without &"a2+ or Mg2+ and dispersed with a magnetic stirrer at room temperature in PBS containing 0.1% collagenase, 0.25% trypsh, 0.82% disodium ethy lenediamine tetraacetic acid (EDTA), and 0. 01. 2% meth- ylcellulose. Harvested cells were decanted. The cell suspen- sions were filtered though several layers of sterile gauze, col- lected by centrifugation (183 x g, 20 min), washed in medium and resuspended in GM1 supplemented with antibiotics (each millilitre contained 200 IU of penicillin, 200 pg of strepto- mycin and 250 pg of gentamicin sulfate). Cells were seeded into replicate 75-cm2 plastic flasks (Coming COT., Coming, NY). Greater than 90% of the beluga cells were viable at the time of cell culture initiation, as detemined by the tnypm blue exclusion method of Hsskins et d. (1956). Cell cultures were incubated at 37OC. After 24 h, medium was replenished. Medium was subsequently replenished weekly (or whenever the pH k c m e acidic) until monolayers eventually became con- fluent ( a b u t 4 wk).

'Thereafter, cells were routinely maintained by application of standard tissue culture procedure (Merchant et d. 1964). Thus,

cells were readily gown at 37OC as monolayer cultures I supplemented with 100 IUmL- ' of penicillin md 1846 - ' of streptomycin, or in GM2 with 50 pgmL- ' gen- sulphate added. The BWK cells were generally sub-

cultaared every 2 to 3 wk at a 1:2 split ratio by dispersal with 0.01% &ypsin4.02% EDTA (Merchant et al. 1964). Supple- mentary stocks sf ealy passage level BWK cells were pre- sewed frozen in enriched medium with 18% dimethyl sulphox- ide in ampules in liquid nitrogen (Shannon and Macy 1973).

For photomicmscopy, cell cultures that were grown on cover- slips were fixed in absolute methanol a d stained with May- Chnwdd-Giemsa stain in the manner described by Merchant et d. (19@),

The BWK cells were tested fss. the presence of mgrcoglasma md bacteria by using the direct culture method (Mycotrim-TC, Hma Media Inc., Berkeley, CA). No evidence of growth by these two contaminants was noted.

Culture flasks containing 76% confluent, actively growing, BWK cells were exposed to medium containing esleemid (0.05 ~ g - d - ') for 6 h. Cells were harvested, fixed, and chsrns- some spreads were prepared for counting according to the me&od of Exley (1975). Solid-stained preparations were made by staining slides in dilute Giemsa fsa 6 nrin. The chromosome modal number was determined for 50 cells at passage level 7 a d again at passage level 14. The sequentidly C-banded ka- yotype was determined in detail for cells at passage level nine. The C-bmds were vvisudized according to the method of Sum- ner (1972). The BWK cell karystype was arranged by using the previously published model for beluga whale by JmelI a d b a s o n (198 1).

Isszyme Electrophoresis

Enzymes were extracted from actively growing B WK cell cultures, BWK cells frozen in mpules, and from mpules (obtained from the ATCC; CCL No. 78) of the Sg 1 K (NBL- 10) cell line from spatted dolphin by the freeze-thaw procedure of Shannon and Macy (1973). The procedwe was modified by increasing the cell concentration 10-fold prior to extraction, and the find medium contained 6). 3 mg.mL- ' d both nicotinamide adenine dinucleotide (NAD) and nicotinhde adenine dim- eleotide phosphate (NADP). A f er cen~fugation (20 000 x g , 30 min) a single crystal of sucrose was added to each (-8.1. d) sample.

Enzymes were extracted from beluga whale kidney tissue by homogenizati,on in a Teflon and glass tissue grinder with m equal volume of 0.25 M sucrose sdudon. Extracts to be used for electrophoresis of glucosephosphate isomerase (GPI) and phssphoglucomutase (PGM) also contained 0.3 rng*mLs" NADP, while extracts to be used for elwtrophsresis of lactate dehydrogenase (LDH) and mdate dehybogenase (MDH) con- tained 0.3 mg-nak-' NAB. Homogenates were clarified by centrifugation at 40 000 x g for 20 min. The supm&mt was diluted 1:IO with sucrose1NAD or NADP solution, prior to electrophoresis.

Starch gel electrophoresis sf the cell culture and tissue extracts was carried out using the semimicro technique of Tsu- yuki et d. ((1966). The electrophoresis buffer system for sep- aration of all isozymes (LDH, MDH, GPI, and PGM) was N- (3-minogropy~)-mo9qsholindci~c acid pH 6. I (Clayton and Tretidc 1972). LDH md MDH isozymes were visualized as described previously (Clayton a d Gee 1969; Clayton et al. 1971). @PI isozymes were stained in a 0.1 M Tris (pH 8.0 HCL) solution containing 1 m g . d - of MgCl, =6H20, 1.5 m g ~ d - ' of fmctose-6-phosphate , 2 u n i t s - d - of glucose- 6-phosphate dehydrogenase, 0.2 n a g - d - ' of NPnHSP, 0.2 mgenal- ' sf nitroblue tetrmoliuw (NBT), md 0.02 m g m L - ' of phenmine methosulphate (PMS). PGM isozymes were stained in a 0.15 M Tricine solution (pH 8 .O NaOH) that also contained 2.5 rng (MgCl,-6H2O)~mL- ' , 5 mg(glucose-1 -phos- phate)-ml- ' , 2 units (glucose-6-phosphate dehydrogen- asel-mL-', 0.5 mg (NADP)mL-', 0.5 rng ( N B T ) - d s r and 0.63 mg (PMS)TI~L-'.

Can. J Fish. Aqmb. Sci., 6/01. 46, 1989

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... .. :p..f..j:; .". .. ..... &3$&, . c.:.:.>. .:<...:.:: %?*,*.X> ....... 54:;$$*. .- . . >2.... ,.,' .......I. :.:....x ,.,-

. !.&& .. ... . , . ....... . & ..... .....

whale kidney (BWK) cells. Passage 9. May-Gmpawald-Giemsa stain. Bar = 40 pm.

Viral Susceptibility Results The following representative may of mammalian vimses was

used to determine the viral susceptibility of BWK cells: Pslio- vims (types I and 31, echovims (27), coxsackieviws (B3 and B5), herpes simplex virus 1, vaccinia virus, influenza virus (A PR8), md simian agent (SA11) which is a rotavirus. The picomavimses were propagated in Buffalo green monkey kid- ney cells (BGMK). When generalized cytspathic effects (CPE) were evident, the infected cell cultures were frozen-thawed (3 x ) to release cell-bound virus, and the %rims-containing fluids were semi-purified by differential centrifugation followed by filtration though 8.45-pm pore diameter filters. The SA1 IL was grown in simian (MA-104) cells and similarly processed, but not filtered before use. The herpesvirus was grown in BGMK cells; whereas the vaccinia vims was propagated in human (HeLa) cells a d the influenza virus in L cells. The latter thee vimses were used as emde vims stocks, preclarified by slow speed centf.ikgatiow at 4°C to remove cellula ddems.

For infectivity assays confluent nm~onolayers of BWM cells (passage levels 15-17) in 25-cm"asks were used. Cells in GM2 containing 50 pgernL-' of gentamicin sulfate were prop agated at 37°C in an atmosphere containing approximately 1% C8,. The media were removed from the cell cultures and the monolayers were inoculated with 0.25 mh, of a vims or with 0.25 mL of GKN (diluent control, consisting of 0.1 % glucose, 0.04% KCl, and 0.8% NaCl). After adsorption for k h, 5 mL sf CM2 was added to each flask as a liquid overlay. The infected cells were incubated at 37°C in a CO, incubator for at least 10 d and observed daily for CPE. If any cytopathslogy was observed, BWK cells were considered presumptively suscep- tible md the cultures were frozen. Later, 8.25 mL of fluid from each of the frozen cu l tws both undiluted and diluted 1: 10-0 was used to inoculate additional monolayers of BWK cells. Cells were reincubated at 37°C and sbsemed daily for CPE. The appearance of CBE indicated that the virus infection was passaged to mother set of BWK cells, md BWK cells were considered to be susceptible t s virus infection and rnultipIication.

The BWK cell cultures contained both epithelioid md fibrs- bbstic ceEl types with the latter apparently pkedominating (See Fig. I). Mature cultures, which grew readily at 337°C in either GMI or GM2, appeared as contact-inhibited cell monolayers that could be transferred (or passaged) by standad tissue cul- ture procedures (Merchant et al. 1964). Cells became increas- ingly granular, however, beyond passage level 17. By passage level 19 2 1 cell growth had reached a plateau md cultures could no longer be propagated in the usual manner. With periodic replenishment of media, these cultures were maintained in the Wimipeg labrapry in a steady-state condition for over a year without transfer. Significantly, cell cultures that had been revived in HonoEamlu from frozen ampules of early passage BWK stock exhibited a similar finite life span (i.e. cells could not be serially propagated beyond passage 20).

The BWK cell line exhibited a diploid mode (2n = 44) when chromosomes were counted at passage levels 7 a d 14. In a detailed hyotype for cells at passage level 9 (Fig. 2) cho- mosomes appeared normal with no evidence sf chromosomal aberrations or overt remmgements, but the distribution sf con- stitutive heterwhromaeiw within some homologous pairs dif- fered very slightly from the published kqo type for this species (Jme1l md Amassn 198 1).

Zyrnograms of Fig. 3 show that GPI (Fig. 3c) and PGM- 1 (Fig. 3 4 isozymes clearly distinguish BWK md Sp B K cells while the mobilities of MDH (Fig. 3a) and LDH (Fig. 3b) iso- zyrnes are the same in both cell lines. In all isszyrne systems the mobilities of the isozymes from BWM cells a d from beluga whde kidney tissue are identical. Although only one electro- phoresis condition (buffer composition, pH etc .) Is represented in Fig. 3 for each isozyrne system, more were tested, and in contrast to the situation with @PI md PGM-I no set of condi- tions was found that would reved different mobilities for BWK and Sp 1 K MDH or LBH.

The BWK cells at passages 15-17 were susceptible to coxsackievims (B3 a d B5), vaccinia, and herpes simplex 1;

1474 Can. J. Fish. A ~ U Q P . Sci., Vob. 46, 1989

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FIG. 2. Beluga whde cell chomosomes (213 = 44). Bar= 110 pm. (A) Solid-stained kaqotype of BWK cells (passage Bevel 9). (B) Sequentially C-banded kayotype.

but were refractory to echovirus (271, influenza A (PR8), polio- vims (types 1 md 3) a d simian agent (SA11). No attempts were made to adapt the culture-negative viruses to BWK cells by serial blind passage of virus beyond passage 2, in ghe manner used by Cabasso et d. (195'7) for adapting infectious bovine rhinotracheitis vims to HeLa cells.

Fibroblastic cell cultures derived from normal tissues of ter- restrid vertebrates usnda8ly have a limited or finite proliferative

capacity, This phenomenon has been most extensively studied in the case of human diploid fibroblasts which generally cannot undergo more than about 50 population douhlings in cell culture (Hayflick 1965; 8968). This aging of cell populations in vitro has k e n correlated by many investigators with senescence sf the whole organism (Macieka-Coelko 1988). Although the in vim proliferative capacity of cells from a given tissue is both species specific and reproducible, there is presently insufficient data to make accurate interspecies comp&sons relating popu- lation doublings of cells in culture to the natural lifespm of the donor animal (Cristofalo et al. 1977).

Cm. 1 Fish. Aquas. S'cS., &%1. 46, 1989 14'75

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6-

1- MMHH

FIG. 3. Zymogrms sf beluga whale tissue and cuIturedj cell enzymes (starch gel electrophoresis at pH 6.1). (a) Malate dehydrogenase (MDH), (b) Lactate dehgrkogenase (LDH), (c) Glucosephosphate issm- erase (GPI)g (dl Bhssphcsglucomutase (PGM), kidney =extract of beluga whale kidney, Sp 1 K = spotted dolphin culture eel1 errtrzict, BWK = beluga whale cultured cell extract.

The beluga BWK cell cultures demonstrated a similar finite lifespan, or proliferative capacity, in both the Winnipeg and Honolulu laborato~es. Whether this represented the natural phenomenon of senescence in vitro referred to previously, or was an undiscovered nutritional deficiency problem, is unclear. Attempts by A n h w s et al. (1973) to grow narwhal cells in MEM with 20% FFB resulted in cultures which were initidly successful, but which ceased to thrive after several celH passages. Similar results were obtained by Smith et d. (1987) who reported that bowhead whale cells grew slowly in vitro, and cultures could not be propagated beyond six to nine passages. These two reports both seem to support the finite lifespan hypothesis. Certainly, cetacean cells are comparatively difficult to culture9 as evidenced by comments in the ATCG catalogue relating to the cultwe of Sp 1 K cells in wonenriched medium (Hay et al. 1983).

R e r e is a general csnsewatism of cbomosomal evolution in cetaceans (Amason 1972). Therefore, c o q ~ s o w s sf kw- yotypes of msnodontid and delphinid cetaceans have focussed on variations in C-hetmochomatin rather than ow overt differ- ences in ckomosome moqhol~gy (Jmell and h a s o n 198 1, h a s o n 1980). Intrapopula~snal differences in C-heterocho- matin distribution have not been extensively studied. In the present study early passage level B cultrases were not sac- rificed for kayological mdyses; instead, efforts were devoted toward obtaining maximum numbers s f cells in culture. Although the kayotype of BWK cells at passage level 9 (Fig. 2) appeared simi2ar to th& for Delphkreapterus leucas published by Jamell and Amson (B981), certain minor changes have likely occurred in the cell cultures with increased cell passage,

as evidenced by our further sksematisn of minor variations in C-banding gat terns between individual B W M cells. Therefore, the finite BWK cell line can best be described as pseudodiploid, rather than diploid, as based upon evidence derived from the sequentially @-banded kayotype (Fig. 2B). Unfortunately, insufficient dividing cells were present in the apparently senes- cent BWK cultures at passage levels 18-20 to allow us to doc- mewt changes occurring later in the terminal cultures.

Isozyrne electrophoresis is a convenient and extensively used means of identifying cell lines (OqBHien et al. 1980). We have not sought to provide a definitive set of isozymes that will dis- tinguish BWK cells from d l other cell h e s brat it is clear (Fig. 3) that GP% md PGM-1 x e sufficient to distinguish BWK and Sp 1 K cells while the electrophoretic mobilities of MBH and LDH isozymes are evidently the same in both of these related species. As well, the same electrophoretic mobilities in extracts from beluga whale kidney and BWK cells for dl the isozyrne systems provides a measure of confidence in their identity.

The vaiety sf vimses which infect marine rn yet been determined. Many unexplained mortalities have occwed in whales and dolphins both in captivity and in the wild that may have had a contributing viral etiology (Britt and Howard 1983). 'To date, diagnostic virology of cetaceans has relied heavily on application sf s t m d d veterinary prscedmes with the supplemental use of cell lines from terrestrial mam- mals. This is due in large p a t to the obvious lack of adequately characterized cetacean cell lines. Apparently only the Sp 1 K line has been tested against a bmad array of viruses, and this test series did not include echovimses, rotavimses, or calici- viruses. Thus, the Sp 1 K cell line was reported to be suscep-

1476 Can. J. Fish. Aquar. Sci., bl. 46, 1989

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tible to ssxsackievims (A9 and B5), herpes simplex, vaccinia, influenza A, parainfluenza 3, reovims 3, adenovims 5, vesic- ular stomatitus virus, infectious bovine rhinotracheitis virus, bovine viral diarrhea agent, hog cholera virus, and a variety of encephalitus viruses (Hay et al. 1983; Kniaaeff and Groyon 1966). The Sp I K cells were refractory only to poliaavims 2 (May et al. 1983). In contrast the fibroblast-like cell lines from the Pacifjc whiteside dolphin were susceptible to coxsackievi- ms types A9, B3, B4, but not to coxsackieviirus types B I , B2, B5, B6 (Cecil md Nigrelli 1970b). These cells were also sus- ceptible to ~ e ~ v i m s (1 and 39 but not adenovims 12. Other viruses were mot tested, so detailed comphsons with the BWK line are not feasible. The stock cultures s f BWK cells in ampules will k~ndoubtedly be useful for additional studies in diagnostic virology.

For heir coopration in allowing us to sample the beluga whales taken during the annual domestic hunt at Clearwater Fjord, we thmk the Inuit hunters sf Bangnirtung, N.W.T. We also thank the personnel of the Department of Fisheries md Oceans, especially Rob Stewart md Jack Om, for their help in the sampling and transporeation of the beluga tissue. Lastly, we thank Grace h c t e r o w for preparation s f the find typed copy. This study was funded in part by the Northern Oil and Gas Action h o g a n (NOGAP), though the Department of Fish- eries and Oceans, Central and Arctic Region, and by U.S. funds from the Office of Naval Research (Contract No. NW14-84-K-0381).

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