Development of a continuous cell line from larval

Received: 29 March 2018

|

Revised: 1 May 2018

|

Accepted: 2 May 2018

DOI: 10.1111/jfd.12830

ORIGINAL ARTICLE

Development of a continuous cell line from larval Atlantic cod (Gadus morhua) and its use in the study of the microsporidian, Loma morhua Michael J. MacLeod1 | Nguyen T. K. Vo2 | Michael S. Mikhaeil1 | S. Richelle Monaghan1 | J. Andrew N. Alexander3 | Mandeep K. Saran3 | Lucy E. J. Lee1,2,3 1 Department of Biology, Wilfrid Laurier University, Waterloo, ON, Canada 2

Department of Biology, University of Waterloo, Waterloo, ON, Canada 3

Faculty of Science, University of the Fraser Valley, Abbotsford, BC, Canada

Abstract In vitro cell culture methods are crucial for the isolation, purification and mass propagation of intracellular pathogens of aquatic organisms. Cell culture infection models can yield insights into infection mechanisms, aid in developing methods for disease mitigation and prevention, and inform commercial-scale cultivation approaches. This

Correspondence Lucy E. J. Lee, Faculty of Science, University of the Fraser Valley, Abbotsford, BC, Canada V2S 7M8. Email: [email protected]

study details the establishment of a larval cell line (GML-5) from the Atlantic cod (Gadus morhua) and its use in the study of microsporidia. GML-5 has survived over 100 passages in 8 years of culture. The line remains active and viable between 8 and 21°C in Leibovitz-15 (L-15) media with 10% foetal bovine serum and exhibits a

Funding information Natural Sciences and Engineering Research Council of Canada, Grant/Award Number: 46159

myofibroblast phenotype as indicated by immuno-positive results for vimentin, asmooth muscle actin, collagen I and S-100 proteins, while being desmin-negative. GML-5 supports the infection and development of two microsporidian parasites, an opportunistic generalist (Anncaliia algerae) and cod-specific Loma morhua. Using GML-5, spore germination and proliferation of L. morhua was found to require exposure to basic pH and cool incubation temperatures (8°C), in contrast to A. algerae, which required no cultural modifications. Loma morhua-associated xenomalike structures were observed 2 weeks postexposure. This in vitro infection model may serve as a valuable tool for cod parasitology and aquaculture research. KEYWORDS

Atlantic cod, cell line, infection model, Loma morhua, microsporidia, xenoma

1 | INTRODUCTION

imposed by an array of pathogens (reviewed in Bricknell, Bron, & Bowden, 2006) and limited tools with which to address them (Som-

The Atlantic cod has a long history as a popular food fish throughout

merset, Krossøy, Biering, & Frost, 2005). If aquaculture programs are

the world (Kurlansky, 1998), supporting large fisheries and, more

to continue their current development, better disease management

recently, commercial-scale aquaculture programs (Badiola, Albaum,

approaches will be required, necessitating research into cod parasites

Curtin, Gartzia, & Mendiola, 2017; Birt, Rodwell, & Richards, 2009;

and cod immunology. Given the market value of live fish and the rel-

Kjesbu, Taranger, & Trippel, 2006; Naylor & Burke, 2005; Rosenlund

ative complexity of in vivo studies, additional research tools are

& Skretting, 2006). Cod farming efforts have been initiated in several

required for timely and efficient means of improving livestock man-

countries on either side of the Atlantic, but continued development

agement and production methods. Cell culture and other in vitro

has been slow, hindered in large part by livestock health challenges

methodologies have proven invaluable tools for studies into fish

J Fish Dis. 2018;1–14.

wileyonlinelibrary.com/journal/jfd

© 2018 John Wiley & Sons Ltd

|

1

2

|

MACLEOD

ET AL.

physiology and parasitology, advancing our understanding of piscine

derived from larval cod (GML-5) and the development of in vitro cul-

pathogens, facilitating the production of vaccines and preliminary eval-

ture methods for the propagation and study of L. morhua.

uations of therapeutic treatments (Bols, 1991; Bols, Pham, Dayeh, & Lee, 2017; Hightower & Renfro, 1988; Lee, Dayeh, Schirmer, & Bols, 2009; McConnachie, Sheppard, Wright, & Speare, 2015; Monaghan et al., 2009; Pinheiro & Bols, 2013; Villena, 2003; Woo & Li, 1990). Cod cell culture reports have been comparatively few and sporadic, yet primary cultures from various tissues have been reported

2 | MATERIALS AND METHODS 2.1 | Establishment and maintenance of the GML-5 cell line

including melanophores (Nilsson, Rutberg, & Wallin, 1996), endocar-

Two-week-old posthatch cod larvae measuring on average 7.20 mm

dial cells (Koren, Sveinbjornsson, & Smedsrod, 1997), head kidney

in length were collected from the University of Maine’s Center for

macrophages

&

Cooperative Aquaculture Research. The larval fish had been reared

Bertheussen, 1997), liver cells (Søfteland, Holen, & Olsvik, 2010),

in 14°C sea water and were fed rotifers ad libitum. Ten larvae were

pituitary cells (Hodne, von Krogh, Weltzien, Sand, & Haug, 2012),

transported on ice packs to Mount Desert Island Biological Labora-

stem cells (Holen, Kausland, & Skærven, 2010) and larval cod

tory, and primary cultures were initiated following several rinses in

(MacLeod et al., 2011). However, availability of stable cod-derived

sterile antibiotic containing sea water as denoted below and prelimi-

cell lines has been scarce. Jensen and Christensen (1981) reported

narily reported by MacLeod et al. (2011).

(Sørensen,

Sveinbjørnsson,

Dalmo,

Smedsrød,

on a cod gonadal cell line that was not preserved, and recently, Jen-

All culture materials and chemicals were purchased from Sigma-

sen et al. (2013) reported on continuous cod cell cultures (Atlantic

Aldrich (St. Louis, MO) unless stated otherwise. Explant cultures of cod

cod larvae [ACL]) that could be available to the research community.

larvae were maintained initially in Leibovitz’s L-15 medium (Hyclone,

These, and the result of the present report, may permit simpler and

Thermo Fisher Scientific) supplemented with 5% filter-sterilized sea

more controlled investigations into various aspects of cod physiol-

water (SSW), 10% foetal bovine serum (FBS), 100 U/ml penicillin and

ogy, toxicology and host–parasite interactions, particularly for obli-

100 lg/ml streptomycin (P/S). The cultures were gradually adapted to

gate intracellular parasites allowing greater understanding of the

L-15 medium without SSW. The regular growth conditions for GML-5

parasites themselves.

included 21°C in L-15 with 10% FBS in free gas exchange, as previ-

In addition to common piscine disease study targets such as

ously indicated with primary cod larval cells (MacLeod et al., 2011).

infectious pancreatic necrosis, viral nervous necrosis and viral haemorrhagic septicaemia, the cod-specific microsporidian Loma morhua represents an ideal subject for in vitro research. Loma morhua is an

2.1.1 | Initiation of larval cod cell cultures

obligate intracellular fungal parasite that causes enormous, spore-

As per MacLeod et al. (2011), primary cultures were initiated by tis-

filled cells known as xenomas (Lom & Dykova, 2005) to form on the

sue explant outgrowth methods similar to those used successfully

gills and intestinal epithelium of afflicted fish (Morrison, 1983), as

for establishing cell lines from other coldwater marine fish species

well as in other internal organs, including spleen and heart (Powell &

such as Pacific herring (Clupea harengus pallasi; Ganassin, Sanders,

Gamperl, 2016). Infections may be widespread in farming operations

Kennedy, Joyce, & Bols, 1999) and haddock (Melanogrammus aeglefi-

(Khan, 2005), causing poor growth, deformations and high mortality

nius; Bryson et al., 2006). All work was performed aseptically under

rates (Khan, 2009). Based on available literature, a schematic depic-

a laminar flow hood and cells were kept at 15°C unless noted other-

tion of the presumptive infective sequence of L. morhua in cod intes-

wise. The Gadus morhua larvae were placed in several sterile 60-mm

tine is outlined in Figure 1.

petri plates in a solution of 29 penicillin–streptomycin–amphotericin

Due to the obligate intracellular nature of microsporidians, cell

(200 U/ml penicillin, 200 lg/ml streptomycin and 0.5 lg/ml ampho-

culture techniques have proven particularly invaluable for propaga-

tericin B) in 0.2 lm filtered SSW. Individual fish were sectioned into

tion of spores (Valencakova et al., 2002) and uncovering aspects of

cephalic, medial and caudal portions and subsequently into 0.1 mm3

the parasite’s lifecycle and physiology (Chen et al., 2009; Gisder,

tissue fragments using a sterilized scalpel and sharp probe. Frag-

€ ckel, Linde, & Genersch, 2011; Leitch & Ceballos, 2008). Much of Mo

ments from each of three body sections per individual larvae were

the work to date, however, has focused principally on arthropod and

transferred into wells of six-well Corning CELLBIND plates and 12-

mammal-infecting microsporidians, with very few fish-infecting spe-

well BD Falcon tissue culture plates, covered with a small amount of

cies represented as in vitro infection models (reviewed in Monaghan

10% FBS L-15 growth media and left undisturbed to allow fragments

et al., 2009). Nonetheless, promising recent reports with fish cell

to adhere. At 1 hr postplating, each well was gently filled with addi-

lines are proving useful for studying fish-infecting microsporidians

tional L-15 media supplemented with varying concentrations of SSW

(McConnachie et al., 2015; Saleh, Kumar, Abdel-Baki, El-Matbouli, &

and FBS, and 19 (v/v) penicillin–streptomycin–amphotericin (100 U/

Al-Quraishy, 2014) or other microsporidians (Monaghan, Rumney,

ml penicillin, 100 lg/ml streptomycin and 0.25 lg/ml amphotericin

Vo, Bols, & Lee, 2011).

B). Cultures were maintained in a 15°C incubator and observed regu-

This study advances the initial report of larval cod cell

larly for cell proliferation. Ten days postplating, successful trials

cultures published by MacLeod et al. (2011), providing further details

(those exhibiting substantial outgrowth) were treated with TryplE

to the establishment and characterization of a continuous cell line

(recombinant trypsin, Gibco, Life Technologies, Burlington, ON,

MACLEOD

ET AL.

FIGURE 1

Schematic sequence of infectivity for Loma morhua in cod intestine. Image © Mike MacLeod, 2012

|

3

4

|

MACLEOD

ET AL.

Canada) and re-plated onto 12.5-cm2 tissue culture flasks (Falcon).

temperature. Secondary antibodies, Alexa Flour 488â-conjugated goat

Most cultures were lost following first few passages but a single trial

anti-mouse or anti-rabbit (Invitrogen) were used in accordance with

from the midsection of larvae 5, in 5% SSW and 10%FBS survived

the primary antibody species source. Secondary antibodies were

and was successfully subcultured afterwards. In response to slow

applied at 1:1,000 dilution for 1 hr. Cells were mounted in DAPI-con-

proliferation following initial passages, cultures were placed at vari-

taining Fluoroshield medium. Fluorescence images were taken with a

ous temperatures, and additional FBS (15%) with or without SSW

Zeiss LSM 510 laser-scanning microscope and confocal images were

supplementation. After several months, stable repeatable growth

acquired and analysed using ZEN lite 2011 software.

was established in L-15 media supplemented with 10% FBS and 1% PS at room temperature (18–21°C).

Alkaline phosphatase (AP) activity has been used as a marker for embryonic stem cells in culture (O’Connor et al., 2008). AP staining

For routine maintenance, GML-5 cell cultures were subcultured at

was performed for GML-5 stem cell potential using the leucocyte

a splitting ratio of 1:2 every 7–14 days. Cell dissociation was per-

AP detection kit (Sigma 85L3R-1KT) following the manufacturer’s

formed with TryplE (Invitrogen). GML-5 was cryopreserved using con-

suggested protocols. Confluent cultures were stained and observed

ventional methods (10% (v/v) dimethyl sulfoxide (DMSO) in growth

against previously identified AP-positive cells (Gignac et al., 2014).

medium and frozen in liquid nitrogen). Cell thawing following cryop-

GML-5 cells were evaluated for cellular senescence as previously

reservation was performed at intervals between several months and

reported for other fish cell lines (Gignac et al., 2014; Vo et al., 2015)

5 years postcryopreservation by rapidly thawing cryovials in tepid

by monitoring senescence-associated b-galactosidase activity using

water, resuspension in culture media, collection of the cell pellets,

the Senescence Cells’ Histochemical Staining Kit (Sigma CS0030).

removal of the supernatant and re-plating the cells in tissue culture flasks. Cell numbers and viability were monitored by Trypan Blue exclusion using hemocytometer-based counting or with automated cell counters (Bio-Rad’s TC-20, or Thermo Fisher’s Countess II).

2.2 | Characterization of GML-5 for species origin identity and cellular attributes

2.3 | Capacity of GML-5 to support the infection and development stages of the opportunistic microsporidian Anncaliia algerae Despite being a principally insect-infecting pathogen, the opportunistic Anncaliia algerae has been found to readily infect cells from a wide variety of host species in vitro—including coldwater fish (Mon-

GML-5 cells at passage 22 were blotted onto FTA cards (Whatman)

aghan et al., 2011). Therefore, it was used to evaluate the suitability

and submitted to the Biodiversity Institute of Ontario (Guelph, ON,

of GML-5 for supporting microsporidian infection and proliferation,

Canada) for “DNA barcoding” (molecular genotyping using cyto-

and facilitate comparative observations of life stages with L. morhua.

chrome c oxidase subunit I or CO1) for species authentication (as

Anncaliia algerae spores were initially obtained from the Ameri-

per Cooper et al., 2007). The resulting CO1 gene sequence was anal-

can Type Culture Collection (Manassas, Virginia; ATCC number PRA-

ysed to match the cell line to originating species using the Basic

168) and propagated in goldfish skin cells (GFSk-S1, Lee, Caldwell, &

Local Alignment Search Tool (BLAST) from GenBank at the National

Gibbons, 1997) as per Monaghan et al. (2011). Anncaliia algerae were

Center for Biotechnology Information (NCBI) database at www.ncbi.

isolated by trypsinizing spore-cell co-cultures, followed by centrifu-

nlm.nih.gov/BLAST as well as the Barcode of Life Data (BOLD) at

gation for 5 min at 300 g. The supernatant was then removed, and

www.barcodinglife.org.

infected cells were lysed in 3 ml of cell-culture-grade water for

Karyotyping, immunofluorescence staining and evaluation for con-

24 hr. The suspension was gently mixed with 3 ml Percollâ and cen-

tinuous cell line characteristics were performed as described for other

trifuged at 1,000 g for 20 min. The supernatant was removed, and

recently established fish cell lines (Gignac et al., 2014; Vo, Mikhaeil,

the pellet of purified spores was suspended in growth media, diluted

Lee, Pham, & Bols, 2015). For karyotyping, metaphase chromosomes

to approximately 4 9 106 spores/ml and inoculated onto confluent

were counted from 82 spreads processed from actively growing GML-

GML-5 cell cultures in 25 cm2 tissue culture flasks. Each culture was

5 cells. Indirect immunofluorescence staining was performed on cells

maintained at room temperature. Phase-contrast microscopy (Nikon

plated in four-chamber tissue culture slides with appropriate controls.

Eclipse TE300) was used for daily inspections to detect the presence

All antibodies tested were previously shown to react to fish antigens

of germinated spores (GS), infected cells, and different intracellular

(Gignac et al., 2014; Vo et al., 2015). Primary antibodies tested

developmental stages of a typical microsporidian. Micrographs were

included: rabbit polyclonal antisalmon collagen I IgG (Cedarlane,

taken using a Nikon Coolpix E990 digital camera.

Burlington, ON), used at 1:400 dilution; mouse monoclonal antiporcine vimentin IgG (clone V9; Sigma-Aldrich), used at 1:200 dilution; mouse monoclonal anti-a-smooth muscle actin (a-SMA) IgG (clone 1A4; Sigma-Aldrich), used at 1:200 dilution; rabbit polyclonal antichicken desmin IgG (Sigma-Aldrich), used at 1:300 dilution; and rabbit polyclonal antibovine S-100 IgG (Sigma-Aldrich), used at 1:200 dilu-

2.4 | Establishing the in vitro infection parameters for L. morhua using GML-5 2.4.1 | Loma morhua spore samples

tion. The anti-vimentin antibody was applied overnight at 4°C,

Samples of mature, infective L. morhua spores were obtained from

although the other antibodies were applied for 1 hr at room

Dr. Michael S. Duffy’s lab at the University of New Brunswick (Saint

MACLEOD

|

ET AL.

John, New Brunswick). Spores were extracted from mature Atlantic cod obtained from nearby aquaculture facilities. Fish exhibiting

5

2.4.4 | Effect of alkaline pH

symptoms of Loma infection—white cysts on gills and spleen—were

Further efforts to stimulate spore germination and infection were

dissected and xenomas were removed, which were subsequently

attempted by simulating the neutral to alkaline pH shift spores are

suspended in 1 ml sterile saline in autoclaved bullet tubes and

subject to upon passage from the stomach to the intestines in a

shipped by courier to the Lee laboratory at Wilfrid Laurier Univer-

prospective host. These conditions were previously found to stimu-

sity. Suspended xenomas were ruptured by vigorous agitation using

late germination in another marine fish-infecting microsporidian

a sterilized sharp probe, and centrifuged to isolate cell debris and

species (Pleshinger & Weidner, 1985). Spores were suspended in

spores from the saline solution. Spores were then re-suspended in

Minimal Essential Medium (MEM) containing 10% FBS. They were

3 ml cell culture-grade H2O, overlaid in 3 ml Percollâ (Sigma) and

then inoculated onto GML-5 cultures in 12.5-cm2 Falcon tissue

centrifuged at 1,200 g for 20 min. Following removal of the super-

culture flasks with 0.5 ml of spore suspension and incubated at

natant, the spore pellet was re-suspended in L-15 growth media sup-

8°C for 1 hr. Without CO2 incubation, MEM pH increases to 8.2

plemented with 29 suggested dose of Gentamicin-Amphotericin B

in the presence of GML-5 cells, facilitating pH shifts while cells

(ThermoFisher, cat. R01510) with final concentrations of gentamicin

and spores primed for germination are in close proximity. Follow-

at 20 lg/ml and amphotericin B at 0.5 lg/ml, to prevent microbial

ing the 1-hr incubation period, flasks were filled with fresh L-15

contamination. The resultant spore suspensions were refrigerated in

media containing 15% FBS, neutralizing the pH and permitting cells

tissue culture flasks prior to inoculation onto GML-5 cells. The

to resume normal cellular functions and metabolism. Co-cultures

L. morhua spore suspensions were positively identified as such, fol-

were observed daily by phase-contrast microscopy for indications

lowing the procedures recently reported by Frenette, Eydal, Hansen,

of infection.

Burt, and Duffy (2017). In brief, PCR was performed using specific primers for L. morhua rDNA internal transcribed spacer sequences (Frenette et al., 2017), which are commonly used for barcoding fungi

2.4.5 | Parameters affecting sporulation MEM trials with slight modifications were conducted to determine

for species identity (Didier, 2005).

whether spore proliferation rates could be enhanced as in other cultured microsporidian species. Prior to resuspension in MEM and

2.4.2 | Preliminary L. morhua infection trials

inoculation onto cells, several spore suspensions were subjected to

Initial infection trials were conducted using two surrogate cell lines

acidic conditions by mixing in L-15 brought to a pH of 5.0 with

and selected for their close phylogenetic relationship with cod

1.0 M HCl. Spores suspended in low pH L-15 were then cen-

(haddock embryos [HEW]; Bryson et al. 2006) and suitable tissue

trifuged and mixed in MEM supplemented with 10 mM MgCl2 and

origin (rainbow trout gills [RTgill-W1]; Bols et al., 1994). These pre-

0.065% mucin (v/v), which was found necessary to initiate spore

liminary trials were performed by inoculating 5 ml of purified

discharge in another piscine microsporidia (Pleshinger & Weidner,

L. morhua

(approximately

1985). 0.5 ml of the spore suspension was then inoculated onto

5

7.3 9 10 spores/ml) onto confluent cultures of surrogate cells in

confluent GML-5 cultures in 12.5-cm2 tissue culture flasks. Plates

2

spores

suspended

in

L-15

medium

tissue culture flasks. Co-cultures were incubated at 8°C

were incubated at both 8 and 18°C. All pH measurements were

and observed using phase-contrast microscopy for signs of spore

made using a Fisher Scientific accumet BASIC AB15 pH meter.

germination, infected cells, and the appearance of developmental

Following the appearance of apparent microsporidial developmental

stages comparable to those observed in the opportunistic A. al-

stages within cells postexposure (PE), infected cultures were pas-

gerae. Further efforts to stimulate successful infection were

saged and observed through a phase-contrast microscope to deter-

attempted by testing the influence of various physical adjustments

mine if spores were viable and if infection had continued

described below.

postpassage.

2.4.3 | Infection-mediating effects of Mg2+

2.4.6 | Spore viability following prolonged storage

Previously found to enhance A. algerae infection in vitro (MacLeod

To test the viability of L. morhua spores following 2 years of storage

et al., 2011), the influence of supplemental MgCl2 was tested for

at 4°C, previously purified spores were used to replicate MEM infec-

stimulatory effects on L. morhua germination and infection. Pelleted

tivity trials. Spore identity was validated as per Frenette et al., 2017.

spores were suspended in L-15 media containing 10% FBS and

Prior to culture inoculation, 1 ml of spore suspension was added to

25-cm

supplemented with 0, 0.1, 1 and 10 mM supplemental MgCl2.

10 ml of sterile milliQ water and centrifuged at 1,000 g for 10 min

0.5 ml of spore suspension was inoculated onto confluent GML-5

at 18°C. The supernatant was discarded, and centrifugation was

12.5-cm2

co-

repeated with 10 ml of milliQ water. The pelleted spores were re-

cultures were incubated at 8°C. Observations for evidence of infec-

suspended in 3 ml of Dulbecco’s modified Eagle medium (DMEM).

tion or spore proliferation were made daily by phase-contrast

Using a haemocytometer, the final suspension concentration was

microscopy.

determined to be approximately 7.3 9 106 spores/ml. One millilitre

cultures

in

Falcon

tissue

culture

flasks,

and

6

|

MACLEOD

of this suspension (7.3 9 106 spores) was added to a confluent 25-cm2 flask of GML-5. As a control, 1 ml of DMEM was added to another 25-cm2 flask of GML-5. Both flasks were incubated at 8°C to mimic in vivo conditions and promote infection and proliferation

ET AL.

3 | RESULTS 3.1 | Cell culture properties Cellular outgrowth was consistently observed from cod larval tissue

of L. morhua. The infection process was monitored through regular inspection

explants, similar to findings reported by Jensen et al. (2013) for ACL

of both the control and experimental flasks. Photomicrographs of

cells. Most adhered explants exhibited minor outgrowth surrounding

infected and control cultures were taken with a Nikon Eclipse

tissue fragments by 24 hr postplating at a range of temperatures

TS100 phase-contrast microscope and a Nikon DS-Fi2 camera at

from 4 to 18°C. Adherent cells spreading around the periphery of

4009 magnification. The media was changed and cells were rinsed

tissue fragments were noticeable within the week (Figure 2a) at

29 with Hank’s balanced salt solution 12 days PE to remove

15°C, and confluent monolayers surrounding primary culture

excess, free-floating spores and allow for easier visualization of

explants were visible after 30 days postplating (Figure 2b). Cells

infected cells. Experimental and control flasks were passaged every

were successfully passaged to new culture vessels and attached cells

2–3 weeks.

with mitotic figures could be seen within 24 hr postpassaging

(a)

(b)

(c)

(d) F I G U R E 2 Phase-contrast micrographs of larval cod cells that gave rise to GML-5. (a) early explant outgrowth after 1 day of culture; (b) confluent monolayer of cells surrounding an explant after 1 month of culture; (c) attached cells after passaging; (d) GML-5 cells maintained at 4°C for 2 weeks. Note mitotic figures (arrowheads). Scale bars = 50 lm

F I G U R E 3 Representative survival and size distribution graph of GML-5 cells after cryopreservation and thawing. Image captured from Countess II automated cell counter for GML-5 cells frozen at passage 25 in July of 2012 and thawed in December of 2017

MACLEOD

|

ET AL.

7

(Figure 2c). Cellular proliferation was noted at temperatures as low

(Accession No HG514359) when BLASTing on the NCBI GenBank

as 4°C (Figure 2d) in L-15 supplemented with 10% FBS. Cells

database. The CO1 gene sequence for GML-5 was deposited in

appeared fibroblastic in shape at low cell densities, becoming epithe-

BOLD within the project “Characterizing Cell Lines from Fish and

lial-like in appearance as cultures reached confluence. Initial growth

Shellfish (CCLF)” as Process ID CCLF051-11. Karyotype analysis (Fig-

following first-passage was extremely slow, and cells originating from

ure 4b) provided a chromosome modal number of 2n = 46, in agree-

most of the explant trials were eventually lost, except for cells

ment with the reported karyotype modal number of 46 for

derived from an explant of larvae 5 that exhibited steady, continued

G. morhua (Ghigliotti et al., 2012). However, only 21% of the

proliferation. These cells were designated as GML-5 for Gadus mor-

counted spreads had the diploid karyotype characteristic of cod and

hua larvae-5.

aneuploid cells with 47 and 48 chromosomes accounted for a quar-

The GML-5 cells have now been subcultured for 8 years, surviv-

ter of the chromosome spreads analysed.

ing over 100 population doublings as well as successful freezing and thawing cycles. Survival rates after freezing in liquid nitrogen ranged from 40 to 95%, depending on cryopreservation length. In the recent

3.2 | Cell line characteristics of GML-5

past, over 60% survival rates were noted after 5 years postfreezing

Although GML-5 cells tested positive at all passages for AP, a mar-

(Figure 3) in a batch of cells frozen at passage 25. These cells were

ker of stem cell-like characteristics (Figure 5a), they were weakly

identical in morphology and behaviour to GML-5 cells from earlier

positive for beta-galactosidase, a marker for senescence (Figure 5b)

and later passages with average cell size in suspension at 16.75 lm

in earlier passages (