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Establishment and characterization of a pronephric stromal cell line (TPS) from rainbow trout,Oncorhynchus mykissW.
Fish & Shellfish Immunology (1995) 5, 441–457
Establishment and characterization of a pronephric stromal cell line (TPS) from rainbow trout, Oncorhynchus m ykiss W. M. L. DIAGO, M. P. LÓPEZ-FIERRO, B. RAZQUIN
AND
A. VILLENA*
Departamento de Biología Celular y Anatomía, Facultad de Biología, Universidad de León, 24071 León, Spain (Received 22 February 1995, accepted in revised form 3 May 1995) A new stromal adherent cell line, called trout pronephric stroma (TPS), was initiated from a long-term pronephric culture of an adult rainbow trout, and subcultured 104 times over a period of 4 years. This study describes the culture conditions and characterization by enzyme-cytochemistry, electron microscopy, isoenzyme profile, cytogenetic techniques, and viral susceptibility. The cell types in TPS cultures consisted of fibroblastic, epithelioid and giant cells. The modal chromosome number is 58. The optimum growth temperature of the TPS cells is 18–22) C, with a population doubling time of 27 h. The TPS line is susceptible to viral haemorrhagic septicaemia (VHS) virus and infectious haemopoietic necrosis (IHN) virus. ? 1995 Academic Press Limited Key words:
cell culture, cell line, pronephros, stromal cells, rainbow trout, fish.
I. Introduction The pronephros of teleosts is an important haemopoietic organ, in which all lines of haemopoietic di#erentiation have been observed, including a considerable capacity for lymphopoiesis (Smith et al., 1970; Zapata, 1979, 1983). Moreover, it has been proposed as a primary lymphoid organ for the production of B cells in salmonids (Kaatari & Irwin, 1985). From ultrastructural (Zapata, 1979) and histochemical (Castillo et al., 1987; Castillo, 1991) studies, the pronephros of O. mykiss has been shown to contain reticular and sinusoidal endothelial cells, the characteristics of which are similar to those of their counterparts in the bone marrow of mammals (Westen & Bainton, 1979) and birds (Yoshida & Yumoto, 1987). Therefore, the trout pronephros would appear to contain haemopoietic microenvironments resembling those of the bone marrow of the higher vertebrates. The techniques of long-term bone marrow cultures (Dexter et al., 1977; Whitlock & Witte, 1987) have provided an excellent methodology for studying the role of haemopoietic microenvironments in mammals (Allen, 1981; Dexter et al., 1990). We have applied these techniques to established long-term cultures of rainbow trout pronephros capable of supporting haemopoiesis (Diago et al., 1993a). From one of such long-term cultures, we have obtained a *Author to whom correspondence should be addressed. 441 1050–4648/95/060441+17 $12.00/0
? 1995 Academic Press Limited
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new stromal adherent cell line, which we call trout pronephric stroma (TPS). The TPS cell line is able to support the development of haemopoiesis when it is co-cultured with haemopoietic precursors (Diago et al., 1993b). In this paper we describe the culture conditions, phenotypical characterization, growth characteristics, isoenzyme profile and viral susceptibility of the TPS cell line.
II. Materials and Methods CULTURE CONDITIONS
The cell line was derived from a healthy adult rainbow trout, Oncorhynchus mykiss (Walbaum), obtained from a commercial fish farm. After anaesthesia with tricaine methane sulphonate (Sandoz), blood was extracted from the caudal sinus and the pronephros was dissected in sterile conditions and placed in 0.16 M phosphate-bu#ered saline (PBS) Ca2+ and Mg2+ free. Long-term cultures were maintained for 8 months (five passages) under the culture conditions described by Diago et al. (1993a). From that time onwards, the TPS cell line was subcultured weekly by trypsinization with 0·05% trypsin–0·02% EDTA solution (Boehringer Mannheim) in PBS, for 3 min at 18&1) C, and 3·5–5#105 cells were transferred to 25 cm2 flasks (Costar) and incubated at 18&1) C in air. At di#erent passages the TPS cell line was cryopreserved, using 1·5 ml Nunc cryovials containing 1–3#106 cells per ml in FCS with 10% dimethylsulphoxide (DMSO). The cells were stored in liquid nitrogen, and cultures were routinely re-established from frozen stocks. The culture medium was RPMI-1640 (BioWhittacker) with L-glutamine and 25 mM Hepes bu#er supplemented with 2 mM L-glutamine (Biochrom), 2·5 mM sodium pyruvate (Merck), 50 ìg ml "1 gentamycin (Biochrom), nucleosides (Sigma)—0·088 mM guanosine, 0·225 mM cytidine, 0·094 mM adenosine, 0·102 mM uridine—, 5#10 "5 M 2-mercaptoethanol (Merck), 10% FCS (Boehringer Mannheim) and 5% allogenic trout serum. After the adjustment of the pH to 7·4–7·6, and the osmolarity to 295·33 mOs kg "1 of water, the medium was sterilized by filtration.
ELECTRON MICROSCOPY
At passage 58 the TPS cells were fixed in situ with cold 2·5% glutaraldehyde in cacodylate bu#er (0·2 M, pH 7·2) for 2 h at 4) C, and post-fixed in 1% osmium tetroxide in the same bu#er for 1 h at 4) C. After two rinses with cold distilled water, cells were scraped o#, pelleted and given consistency using 2% agar. The pieces were then dehydrated in acetone, contrasted with 1% uranyl acetate in 70% acetone and embedded in araldite (Durcupan ACM). Semithin and ultrathin sections were obtained with an Ultracut-E ultratome (Reichert-Jung). The semithin sections were stained with a 1% aqueous solution of toluidine blue in borax to select appropriate areas. Ultrathin sections were counterstained with lead citrate (Reynolds, 1963) and examined with a JEOL EM-1010 electron microscope at 60 Kv.
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Table 1. Monoclonal (m) and polyclonal (p) antibodies used in phenotyping the TPS cell line Primary antibodies Anti-Keratin (p) Anti-S100 (p) Anti-Collagen I (p) Anti-Collagen III (p) Anti-Vimentin Clon VIM3B4 (m)
Specificity
Source
Bovine pan-Keratin Bovine S100 protein Human & Bovine Collagen I Human & Bovine Collagen III Bovine Vimentin
Dakopatts Sigma Southern. Biotech. Assoc.
Mouse Ig Rabbit IgG Goat Ig
Dakopatts Sigma Dakopatts
Southern. Biotech. Assoc. Boehringer Mannheim
Secondary antibodies Rabbit anti-mouse Ig Goat anti-rabbit Ig Rabbit anti-goat Ig
CYTOCHEMICAL ANALYSIS
At passages 14, 34, 60 and 82, the following enzyme activities were assayed: acid phosphatase (ACPH) (Barka & Anderson, 1962), alkaline phosphatase (AKPH) (Burstone, 1958) and non-specific alpha-naphthyl acetate esterase (ANAE) (Pearse, 1972). Negative controls for the specificity of the enzyme reactions were established using incubation media in which the corresponding substrates were lacking. IMMUNOCYTOCHEMICAL ANALYSIS
At the same passages indicated above, primary antibodies against markers of specific mammalian cell types and of connective tissue fibres, indicated in Table 1, were used to characterize the TPS cell line by means of the indirect immunoperoxidase method. Endogenous peroxidase was blocked with 0·3% H2O2 in methanol for 10 min before incubation with the primary antibodies. TPS cultures were incubated for 1 h with appropriate dilutions of the monoclonal or polyclonal antibodies, and then incubated for 30 min with appropriate solutions of peroxidase-conjugated antibodies in 0·16 M PBS (Table 1). The peroxidase reaction was developed with 3,3-diaminobenzidine tetrahydrochloride (Sigma) 0·5 mg ml "1 in Tris-HCl 0·05 M, pH 7·6, plus 0·03% H2O2. Positive controls were carried out on cryostat tissue sections from the mammalian species against which the primary antibodies were produced, and from trout kidney, spleen, thymus, skin and gut. Negative controls for specific immunostaining of the TPS cell line, as well as trout and mammalian tissues, were performed by omitting the primary antibody. PHAGOCYTIC ASSAY
At passage 34, a phagocytic assay was performed by incubating TPS cultures for 1 h at 18&1) C with heat-inactivated yeast (Candida lipolytica)
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stained with neutral red (0·5% in 96% ethanol). A total of 200 cells per flask were counted, and the percentage of cells with yeasts engulfed in their cytoplasm was then calculated.
CYTOGENETIC TECHNIQUES
Karyotype Karyotype analyses of the TPS cell line (at passages 6, 16, 28, 42, 60, 62, 74) were performed as described by Worton & Du# (1979). The chromosomes were stained with a 4% Giemsa solution for 10–15 min, and spreads of 100 cells were counted. NOR staining Nucleolar organizer regions (NORs) were silver-stained by Gold & Ellison’s method (1982).
ISOZYME ANALYSIS
Isozyme profiles at passage 74 were generated using horizontal starch gel electrophoresis and the specific enzymes assayed as described by Taggart et al. (1981). Cells were homogenized in an equal volume of PTP [0·05 M Pipes (Sigma), 0·05% Triton X-100 (Sigma), 0·2 mM Pyridoxal-5*-phosphate (Sigma), pH 6·8]. Gels were assayed for the following enzymes: adenylate kinase (AK), alcohol dehydrogenase (ADH), aspartate aminotransferase (AAT), creatine kinase (CK), fructose 1, 6 diphosphatase (FDP), fumarase (FUM), glucose-6phosphate dehydrogenase (G-6-PDH), glycerol-3-phosphate dehydrogenase (G-3-PDH), hexokinase (HK), isocitrate dehydrogenase (IDH), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), malic enzyme (ME), phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (6-PGDH), phosphoglucose isomerase (PGI), phosphomannose isomerase (PMI), sorbitol dehydrogenase (SDH). Isozyme profiles of the TPS cell line were compared with those from the following cell lines: RTG-2 (from gonad tissue of rainbow trout), CHSE-214 (from Chinook salmon embryo), and BF-2 (from bluegill fry caudal trunk).
CELL GROWTH
Rate of growth The growth of the TPS cell line (at passage 65) was analysed in triplicate as described by Freshney (1987). Briefly, 105, 3#104 and 104 cells per well in 1 ml were seeded into 24-well plates (Costar). In addition, 1·6#104, 4·8#103 and 1·6#103 cells per well in 200 ìl were seeded into 96-well plates (flat-bottom) (Costar). In all cases, cells were incubated at 18&1) C. Cells from three wells (24-well plates), and from 12 wells (96-well plates) were daily removed using trypsin/EDTA and the cell number counted using a haemocytometer. Cell viability was calculated by the trypan blue exclusion method.
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Optimum growth temperature To determine the optimum growth temperature 24-well plates were seeded with 3#104 cells per well in 1 ml, and incubated at 4, 10, 12, 18 and 22) C. The experiment was done in triplicate and extended over 15 days. Cells were harvested daily, counted, and the cell viability determined as described above. 3
H-thymidine incorporation
Rates of DNA synthesis by incorporation of 3H-thymidine were determined in duplicate in 96-well plates (flat-bottom) over 20 days. TPS cells (at passage 65) were plated at 1·6#104, 4·8#103 and 1·6#103 cells per well in 200 ìl and incubated at 18&1) C. Cultures were pulsed with 10 ìl of a 50 ìCi ml "1 3 H-thymidine solution (DuPont) that was added to each culture well. Cells were harvested 24 h later by water lysis onto glass-fibre filters using a semi-automatic cell harvester (Skatron, Norway). Filter discs were dried for 30 min at 80) C, placed into scintillation vials, and 5 ml of scintillation cocktail (Biogreen-1, Scharlau) was added per vial. The number of disintegrations per minute (DPM) were calculated using a calibrated scintillation counter (Beckman LS 6000 TA). SUSCEPTIBILITY TO VIRUSES
Confluent cultures in 25 cm2 flasks were infected with the fish viruses causing infectious haemopoietic necrosis (IHN-V Cedar strain), infectious pancreatic necrosis (IPN-V strain 299), and viral haemorrhagic septicaemia (VHS-V strain 0.7.71), at multiplicities of infection (Basurco & Coll, 1989) ranging from 0·001 to 0·5. The infections were performed as previously described (Diago et al., 1993), and the infected cultures were incubated at 14) C. They were observed with an inverted microscope (Nikon) to determine the occurrence of a cytopathic e#ect. Parallel control uninfected cultures were maintained in the same conditions. III. Results From the earlier subcultures, the TPS cell line comprised adherent cells, mainly showing fibroblastic morphologies. These cells did not show the contact inhibition property, as they did not form true monolayers, some growing over others, even before reaching confluence in the flask. After 104 passages, the TPS cell line has maintained these characteristics. MORPHOLOGY OF THE ESTABLISHED TPS CELL LINE
By light microscopy, initially after a passage, the TPS cells grew forming cell clusters, in which large cells, with multilobulated nuclei, appeared surrounded by fibroblastic cells [Fig. 1(a)]. Later on, TPS cultures did not form true monolayers, but the cells overlapped each other in a multilayered network [Fig. 1(b)]. Confluent TPS cultures consisted of three morphologically di#erent adherent cell types: (a) Elongated or fusiform fibroblastic cells, with
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Fig. 1. Phase contrast micrograph of TPS cultures. (a) 3-day-old culture containing a giant cell (G) and fibroblastic cells (]), #199; (b) general aspect of a 1-week-old culture formed by overlapping fibroblastic (]) and epithelioid (x) cells. Note the giant cell (G), #274; (c) detail of a 1-week-old culture showing the characteristics of the epithelioid cells (x), #364; (d) detail of a 1-week-old culture in which a giant cell (G) is partially overlapped by fibroblastic cells (]), G indicates cell in mitosis, #366.
ovoid nuclei and filiform processes [Fig. 1(b)] formed the major component of the culture; (b) Epithelioid cells, with flattened prismatic cell bodies, which sometimes contained numerous small vesicles, and round nuclei [Figs 1(b) & (c)]; and (c) A low number of giant cells [Figs 1(b) & (d)], resembling those found in the early cell clusters [Fig. 1(a)], with very large rounded cell bodies. Cells of this type showed apparently multilobulated nuclei [Fig. 1(a)] or were multinucleated, most of their organelles, which included clear vesicles, appearing clustered around the nuclear area [Fig. 1(d)]. Ultrastructurally, the cultures consisted mainly of several overlapping layers of elongated cells, with intercellular spaces partially filled by filamentous material [Fig. 2(a)]. The fibroblastic cells had tapered ends and large
Fig. 2. Ultrastructure of the TPS cell line. (a) Multilayered fibroblastic cells. Nucleus (N), rough endoplasmic reticulum (]), multivesicular body (c), note the filamentous extracellular material (*) and the cell-cell contacts ( ), #12 000; (b) higher magnification of the cytoplasm of fibroblastic cells showing elongated (]) and dilated cisternae (G) of rough endoplasmic reticulum, and microfilaments (c), note the extracellular matrix (*), nucleus (N). #30 000; (c) micrograph showing two epithelioid cells (E), nucleus (N), Golgi apparatus (]), and the characteristic vesicles (G), #8000; (d) higher magnification of the cytoplasm of an epithelioid cell showing microfilaments (c) and a desmosome ( ), #25 000.
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Fig. 3. Ultrastructure of the TPS cell line. (a) Micrograph showing part of a giant cell (G) and two epithelioid cells (E). Note two nuclei (N1-N2) of the giant cell. Golgi apparatus (]), #10 000; (b) high-power magnification of the fibrillar material present in the extracellular spaces, fibroblastic cell (F), #40 000.
slender cell branches. They exhibited fusiform nuclei with a band of perinuclear heterochromatin, and one or more nucleoli [Fig. 2(a)]. Major cytoplasmic organelles were numerous free ribosomes, and a well developed rough endoplasmic reticulum, formed by elongated and dilated cisternae containing a material of moderate electron density [Fig. 2(b)]. Some mitochondria, dictiosomes of the Golgi apparatus, short bundles of microfilaments, multivesicular bodies, and some dense bodies were also observed [Figs 2(a) & (b)]. Although there were close appositions of the cell membranes of neighbouring fibroblastic cells [Fig. 2(a)], no cell junctions were observed between them, or with other cell types. Epithelioid cells were characterized by their lobulated, irregular, euchromatic nuclei, and large cytoplasms [Fig. 2(c)], which contained a very well-developed Golgi apparatus, some rough and smooth endoplasmic reticulum, and numerous mitochondria of high electron density [Figs 2(c) & 3(a)]. The presence of numerous, large, electron-clear vesicles, sometimes containing heterogeneous material, was a characteristic marker of the epithelioid cells [Figs 2(c) & 3(a)]. They also contained microfilaments, and were occasionally joined to one another by desmosome junctions [Fig. 2(d)]. Giant cells were very di$cult to recognize in the ultrathin cross-sections, due to the fact that they extend over long, but narrow, areas of the
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Table 2. Cytochemical patterns of the TPS cell line (passage 60 and 82; the latter is in brackets) AKPH
ACPH
ANAE
"(") "(") "(")
"(") "(+/") "(+/")
"(+/") "(+/") + +/+(+ +/+)
Fibroblastic cells Epithelioid cells Giant cells
multilayers. They showed ultrastructural features similar to those described for the epithelioid cells, but with more lobulated and irregular nuclei, and sometimes appearing multinucleated [Fig. 3(a)]. The intercellular substance between the cells [Fig. 2(a) & (b)] was formed by amorphous materials and some striated fibrils [Fig. 3(b)]. CYTOCHEMICAL ANALYSIS
Controls for the enzyme activities assayed were always negative. The cytochemical features of the cell types found in the TPS cultures (passages 60 and 82) are summarized in Table 2. IMMUNOCYTOCHEMICAL ANALYSIS
Positive controls confirmed that the primary antibodies to mammalian markers cross-reacted with components in the trout tissues. However, TPS cells did not stain with antibodies to mammalian keratin, collagen types I and III, vimentin and S-100 protein. PHAGOCYTIC ASSAY
After 1 h of co-culture of TPS cell line and C. lipolytica, only 1% of the cells had engulfed yeasts. CYTOGENETIC TECHNIQUES
Chromosome counts Chromosome analyses showed the TPS cells to be heteroploid. They contained from 46 to 66 chromosomes, more metacentric (42–50) than acrocentric (10–18), which showed a gradual variation in size. The modal chromosome number of the TPS cell line found at passage 60 was 58. Frequency distribution of chromosomes in 100 cells is shown in Fig. 4. Ag-NOR staining Two NOR-bearing chromosomes were seen in all silver-stained metaphases (Fig. 5). This chromosome pair was submetacentric, with the NOR located in the long arms of the two chromosomes.
M. L. DIAGO ET AL.
450 30
25
Cell number
20
15
10
5
0 44 46 48 50 52 54 56 58 60 62 64 66 68 Chromosome number
Fig. 4. Frequency distribution of chromosomes in TPS cells per 100 cells.
Fig. 5. Metaphase chromosomes of the TPS cell line stained for demonstration of NOR-bearing chromosomes (]), #1719.
ISOZYME ANALYSIS
The TPS cell line was positive for the isozymes shown in Table 3. In addition, in these assays the relative electrophoretic migration of each isozyme was used to di#erentiate the TPS cell line from other cell lines (Table 3). Thus, the four cell lines could be di#erentiated by the isozyme profiles for the malate dehydrogenase (MDH). Moreover, the TPS cell line could be
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Table 3. Comparison of the isozyme profiles between the TPS cell line and other fish cell lines Isozyme
TPS v. BF-2
TPS v. RTG-2
TPS v. CHSE-214
AK CK FDP HK IDH LDH MDH 6-PGDH PGI PGM PMI
1 1 3 1 1 1 1 1 2 1 2
2 1 3 2 1 2 1 2 2 1 3
2 3 2 3 3 2 1 2 2 3 3
1, di#erent profile; 2, identical profile; 3, detectable activity in the TPS cell line but no activity in the other cell lines.
di#erentiated from the BF-2 cell line by the AK, CK, HK, IDH, LDH, 6-PGDH, and PGM isozymes and from the RTG-2 cell line by the isozyme profiles for the CK, IDH and PGM. The other seven enzymes assayed were not suitable for di#erentiating these fish cell lines, because they lacked detectable activity in the majority of the lines or had blurred profiles.
CELL GROWTH
TPS cells are relatively easy to maintain as adherent cultures in plastic flasks or dishes. Attempts to grow TPS cell line in suspension cultures have been unsuccessful. Rate of growth The growth curves of the TPS cell line (passage 65) at 18) C, in 24-well and 96-well plates are represented in Figs 6(a) & (b) respectively. The population doubling time, calculated from the middle of the exponential phase of growth for the initial cellular density of 3#104 cells ml "1 (1·5#104 cells per cm2), was 27 h. Optimum growth temperature Growth curves for the TPS cell line cultured at di#erent temperatures are shown in Fig. 6(c). At 10) C or 12) C no increase in cell numbers was observed for 15 days. In the range of temperature tested, optimum growth was at 18–22) C. At 4) C the cells remained viable and cell growth continued very slowly over long periods of time. In this case, TPS cells maintained in 25 cm2 flasks could be subcultured once a year.
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452 (a)
(b) 5
6
10
Cell number
10
104
105
103
104 0 2 4 6 8 10 12 14 16 18 20 22
0 2 4 6 8 10 12 14 16 18 20 22
(c) 5
Cell number
10
104
103
0
4
6
8 10 12 14 16 Days
Fig. 6. Growth curves of TPS cells. (a) In 24-well plates (4) 105 c/w, (/) 3#104 c/w, (-) 104 c/w; (b) in 96-well plates (4) 1·6#104 c/w, (/) 4·8#103 c/w, (-) 1·6#103 c/w; (c) at various temperatures (;) 22) C, (4) 18) C, (/) 12) C, (-) 10) C. c/w (cells per well) indicates starting seeding densities. Each point represents the mean of three counts& S.D. 3
H-thymidine incorporation At a seeding density of 1·6#104 cells per well (5#104 cells per cm2) maximal 3 H-thymidine incorporation was reached after 6 days in culture (Fig. 7). This peak corresponds with the increase in the cell number which was observed between days 6 and 8 of culture [Fig. 6(b)]. At a seeding density of 4·8#103 cells per well (1·5#104 cells per cm2), maximum 3H-thymidine incorporation was reached after 12 culture days. At a seeding density of 1·6#103 cells per well (5#103 cells per cm2) there was a continuous increase in the incorporation of 3H-thymidine for the duration of the experiment. SUSCEPTIBILITY TO VIRUSES
VHS and IHN viruses, produced a cytopathic e#ect (CPE) but IPN-V did not. The CPE began with detachment of scattered cells after 3–5 days. At 7 days post-infection or later depending on the multiplicity of infection, infected cultures showed small areas devoid of cells. Cultures were totally lysed by three weeks post-infection with VHS-V and IHN-V regardless of the initial multiplicity of infection used.
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DPM
2 × 105
1 × 105
5 × 104
4 × 102
0
2
4
6
8
10 12 14 16 18 20 22 Days
Fig. 7. Kinetics of 3H-thymidine incorporation in TPS cells grown at 18) C. c/w (cells per well) indicates starting seeding densities. Each point represents the mean of two rows (n=24)& S.D. (4) 1·6#104 c/w, (/) 4·8#103 c/w, (-) 1·6#103 c/w.
IV. Discussion Although there are in vitro studies concerning the immunological capacity of the fish pronephros, and some kidney stromal cell lines have been established (Chen & Kou, 1987; Tung et al., 1991), no special attention has been paid to the development of long-term pronephric cultures, which may support in vitro haemopoiesis. Recently, long-term cultures from the pronephros of O. mykiss, which are able to support in vitro haemopoiesis have been developed (Diago et al., 1993a; Siegl et al., 1993). From one of such long-term pronephric cultures started in 1990, we have established the TPS cell line described here. The successive subcultures eventually lost the haemopoietic capacity, and became formed only of stromal cell types. The TPS cell line is comprised of fibroblastic, epithelioid and giant cell populations, distinguishable by their morphology and cytoenzyme profiles. These cell types grow in culture forming multilayered networks, which resemble those found in the long-term haemopoietic cultures of the pronephros (Diago et al., 1993a; Siegl et al., 1993). This kind of arrangement is also similar to that observed in long-term cultures of mammalian bone marrow, and the zones where cultured stromal cells arrange themselves tridimensionally are likely to be sites of haemopoiesis (Spooncer & Dexter, 1984). This features of the TPS cell line could explain its ability to support the development of haemopoiesis when it is co-cultured with haemopoietic precursors (Diago et al., 1993b). Stromal cell types similar to the fibroblastic and to the epithelioid cells observed in the TPS cell line are also present in the long-term haemopoietic cultures of the pronephros (Diago et al., 1993a; Siegl et al., 1993). The fibroblastic cells resemble the reticular cells which form the stroma of the
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pronephros in vivo. However, the fibroblastic cells of the TPS cell line lack alkaline phosphatase activity, an enzyme marker of reticular cells of the trout pronephros (Castillo et al., 1987), and also of the mammalian (Westen & Bainton, 1979; Bainton et al., 1986) and bird (Yoshida & Yumoto, 1987) haemopoietic bone marrow. This activity has been associated with the secretion by these cells of either elements of the tissue matrix (ground substance and connective fibres) or haemopoietic factors (Landreth et al., 1981; Song et al., 1985; Dorshkind, 1987). In this respect, there occurred masses of amorphous and fibrillar materials in the intercellular spaces of the cultures. The morphology of the fibrillar material resembles that of the reticulin fibres, thus suggesting that the TPS cell line is able to release components of the connective tissue matrix. This was also true of the long-term haemopoietic cultures of the pronephros (Diago et al., 1993a). However, immunostaining for collagen types I or III in the TPS cultures was negative, which may indicate that the collagen-like fibres are not of these collagen types or, alternatively, that there occurred a defective collagen synthesis in vitro, which resulted in the loss of its antigenic properties. The epithelioid and giant cells show similar structural features and cytoenzyme profiles. From our observations, we think that giant cells may di#erentiate from epithelioid cells. We also observed epithelioid cells in long-term pronephric cultures (Diago et al., 1993a), and we related them to the sinusoidal endothelial cells. The presence of multinucleate giant cells has been described in long-term pronephric cultures (Siegl et al., 1993), and this fact has been related to the ability of macrophages to fuse in culture (Secombes, 1985). Nevertheless, we have not observed fusion of epithelioid cells in the TPS cultures. Although the epithelioid and giant cells show ANAE activity, an enzyme marker of trout macrophages (Razquin et al., 1990), there are no melanomacrophages or typical macrophages in the TPS cell line. Nor is there any remarkable phagocytic activity. Moreover, the epithelioid and giant cell types were always negative for all the cell immuno-markers we have tested, and so their cell lineage remains obscure. The TPS cell line seems to remain cytogenetically stable through the passages, as demonstrated by classical karyotyping. The type and modal number of chromosomes of the cell line are in the range of the values described for rainbow trout (Thorgaard, 1976; Hartley, 1987). Moreover, our results from the selective staining of the NORs agree with those reported for the normal diploid karyotype of rainbow trout (Phillips & Ihssen, 1985; Lozano et al., 1992). Isoenzyme analysis demonstrated that the malate dehydrogenase (MDH) isozyme profile is useful to distinguish the fish cell lines studied here. The profiles for IDH isoenzyme also demonstrated di#erences between the TPS and the RTG-2 cell lines. Growth at various incubation temperatures was evaluated in several experiments to determine the range of growth temperatures as well as the optimum growth temperature for TPS cells. Like other salmonid cell lines (Gravell & Malsberger, 1965; Wolf & Quimby, 1969; Bruce & Byrne, 1973), TPS cells are capable of growth throughout a temperature range from 4) C to 22) C, with maximum growth at 18) C to 22) C. Determination of rate of DNA synthesis by
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incorporation of 3H-thymidine demonstrated that a detectable activity remained for the duration of the experiment. This residual incorporation rate is probably due to (a) the ability of the cells to grow in multilayers, and (b) to the replacement of dead cells, and confirms that there is no contact inhibition in the TPS cell line. The susceptibility of the TPS cell line to IHN-V and to VHS-V agrees with that found for the pronephric stromal cells to VHS-V (Diago et al., 1993), thus indicating that this property has been retained throughout the successive passages. This feature would allow the use of the TPS cell line to analyse the pathogenic mechanisms of these viral agents, as well as for diagnostic purposes. On the other hand, haemopoiesis is regulated by a delicate balance between cell-cell interactions and a network of numerous growth factors, some of them released by blood cells, and others by stromal cells (Dorshkind, 1990). This network of cytokines and growth factors is probably also present in fish (Graham & Secombes, 1990), and cell-cell contacts between developing cells and the stromal cells appear to be necessary for trout haemopoiesis (Diago et al., 1993a). The TPS cell line, which seems to maintain the cellular components and spatial relationships found in the main haemopoietic tissue of trout, may represent a useful tool for studying the mechanisms regulating fish haemopoiesis. This work has been partially funded by the Spanish CICYT, project No. MAR-910851, and the III Framework Programme of the EU, Contract AIR1 CT92 0036. The authors thank Dr P. Martínez (Universidad de Santiago, Lugo, Spain) for the technical assistance with the isozyme analyses. Viral infections were done at the high biological security laboratory (P3) of the INIA-CISA (Madrid, Spain), under the supervision of Dr J. Coll. We also thank the ‘Piscifactoría Los Leoneses’ for providing rainbow trout. M. L. Diago holds a fellowship from the Diputación Provincial de León.
References Allen, T. D. (1981). Haemopoietic microenvironments in vitro: ultrastructural aspects. In Microenvironments in haemopoietic and lymphoid differentiation (R. Porter & J. Whelan, eds), pp. 38–67, Ciba Foundation Symposium 84. London, Pitman. Bainton, D. F., Maloney, M. A., Patt, K. M. & Stern, R. (1986). Characterization of rabbit stromal fibroblast derived from red and yellow bone marrow. Journal of Experimental Medicine 163, 400–413. Barka, T. & Anderson, P. J. (1962). Histochemical methods for acid phosphatase using hezazonium pararosanilin as coupler. Journal of Histochemistry and Cytochemistry 10, 741–753. Basurco, B. & Coll, J. M. (1989). Spanish isolates and reference strains of viral haemorrhagic septicaemia virus show similar protein size patterns. Bulletin of the European Association of Fish Pathologists 9, 92–95. Bruce, L. N. & Byrne, C. (1973). An established cell line from the Atlantic salmon (Salmo salar). Journal Fisheries Research Board Canada 30, 913–916. Burstone, M. S. (1958). Histochemical comparison of naphthol AS-phosphates for the demonstration of phosphatases. Journal of the National Cancer Institute 20, 601–615. Castillo, A. (1991). Ontogenia del sistema inmunitario de la trucha arco iris, Oncorhynchus mykiss. Analisis estructural, inmunocitoquímico y funcional. Ph D Thesis. University of León. Spain.
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Castillo, A., Razquin, B., López-Fierro, P., Alvarez, F., Zapata, A. & Villena, A. J. (1987). An enzyme-histochemical study of the stromal cells and vascularization of the lymphoid organs of the rainbow trout, Salmo gairdneri Richardson. Cuadernos Marisqueros Publicación Técnica 12, 167–172. Chen, S. N. & Kou, G. H. (1987). Establishment characterization and application of 14 cell lines from warm-water fish. In Invertebrate and Fish Tissue Culture (Y. Kuroda, E. Kurstak & K. Maramorosch, eds), pp. 218–227. Tokyo: Japan Scientific Society Press. Dexter, T. M., Allen, T. D. & Lajtha, L. G. (1977). Conditions controlling the proliferation of haemopoietic stem cells in vitro. Journal of Cell Physiology 91, 335–344. Dexter, T. M., Coutinhot, L. H., Spooncer, E., Heyworth, C. M., Daniel, C. P., Schiro, R., Chang, J. & Allen, T. D. (1990). Stromal cells in haemopoiesis. In Molecular Control of Haemopoiesis (eds), pp. 76–95, Ciba Foundation Symposium 148. Chichester: Wiley. Diago, M. L., Estepa, A., López-Fierro, M. P., Villena, A. & Coll, J. M. (1993). The in vitro infection of the hemopoietic stroma of trout kidney by hemorrhagic septicemia rhabdovirus. Viral Immunology 6, 185–191. Diago, M. L., López-Fierro, M. P., Razquin, B. & Villena, A. (1993a). Long-term myelopoietic cultures from the renal haemopoietic tissue of the rainbow trout, Oncorhynchus mykiss Walbaum: phenotypic characterization of the stromal cells. Experimental Hematology 21, 1277–1287. Diago, M. L., López-Fierro, M. P., Razquin, B. & Villena, A. (1993b). Long-term hemopoietic cultures from the pronephros of rainbow trout, Oncorhynchus mykiss Walbaum. In Workshop on Intra- and Extra-Cellular Signalling in Hematopoiesis (Centre for International Meetings on Biology. Instituto Juan March de Estudios e Investigaciones, eds) 16, p. 61. Madrid. Dorshkind, K. (1987). Bone marrow stromal cells and their factors regulate B cell di#erentiation. Immunology Today 8, 191–193. Dorshkind, K. (1990). Regulation of hemopoiesis by bone marrow stromal cells and their products. Annual Review of Immunology 8, 111–137. Freshney, R. I (1987). Culture of Animal Cells. A Manual of Basic Technique. (A. R. Liss, ed), pp. 238–241. New York: Wiley-Liss. Gold, J. R. & Ellison, J. R. (1982). Silver staining for nucleolar organizing regions of vertebrate chromosomes. Stain Technology 58, 51–55. Graham, S. & Secombes, C. J. (1990). Cellular requirements for lymphokine secretion by rainbow trout Salmo gairdneri leucocytes. Development and Comparative Immunology 14, 59–68. Gravell, M. & Malsberger, R. G. (1965). A permanent cell line from the fathead minnow (Pimephales promelas). Annals of the New York Academy of Sciences 126, 555–565. Hartley, S. E. (1987). The chromosomes of salmonid fishes. Biological Reviews 62, 197–214. Kaattari, S. L. & Irwin, M. J. (1985). Salmonid spleen and anterior kidney harbor populations of lymphocytes with di#erent B cell repertoires. Developmental and Comparative Immunology 9, 433–444. Landreth, K. S., Rose, C. & Clagget, J. (1981). Myelogenous production and maturation of B lymphocytes in the mouse. Journal of Immunology 127, 2027–2034. Lozano, R., Ruíz Rejón, C. & Ruíz Rejón, M. (1992). A comparative analysis of NORs in diploid and triploid salmonids: implications with respect to the diploidization process occurring in this fish group. Heredity 69, 450–457. Pearse, A. G. E. (1972). Histochemistry: Theoretical and Applied. Vol. 2, 3rd edition. Edinburgh: Churchill Livingstone. Phillips, R. B. & Ihssen, P. E. (1985). Chromosome banding in salmonid fish: nucleolar organizer regions in Salmo and Salvelinus. Canadian Journal of Genetics and Cytology 27, 433–440.
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Razquin, B. E., Castillo, A., López-Fierro, P., Alvarez, F., Zapata, A. & Villena, A. J. (1990). Ontogeny of IgM-producing cells in the lymphoid organs of rainbow trout, Salmo gairdneri Richardson: an immuno- and enzyme-histochemical study. Journal of Fish Biology 36, 159–173. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Stain Technology 35, 313–323. Secombes, C. J. (1985). The in vitro formation of teleost multinucleate giant cells. Journal of Fish Diseases 8, 461–464. Siegl, E., Albrecht, S. & Lüdtke, B. (1993). Long-term liquid culture of haematopoietic precursor cells from the head kidney and spleen of the rainbow trout (Oncorhynchus mykiss). Comparative Haematology International 3, 168–173. Smith, A. M., Wivel, N. A. & Porter, M. (1970). Plasmacytopoiesis in the pronephros of the carp (Cyprinus carpio). Anatomical Record 167, 351–370. Song, Z. X., Shadduck, R. K., Innes, D. J., Waheed, A. & Quesenberry, P. J. (1985). Hematopoietic factor production by a cell line (TC-1) derived from adherent murine marrow cells. Blood 66, 273–281. Spooncer, E. & Dexter, T. M. (1984). B Long-Term Bone Marrow Cultures. Bibliotheca Haematologica 48, 366–383. Taggart, J., Ferguson, A. & Mason, F. M. (1981). Genetic variation in Irish populations of brown trout (Salmo trutta L.): electrophoretic analysis of allozymes. Comparative Biochemistry and Physiology 69B, 393–412. Thorgaard, G. H. (1976). Robertsonian polymorphism and constitutive heterochromatin distribution in chromosomes of the rainbow trout (Salmo gairdneri). Cytogenetics and Cell Genetics 17, 174–184. Tung, L. C., Chen, S. N. & Kou, G. H. (1991). Three cell lines derived from spleen and kidney of black porgy Acanthopagrus schlegeli. Gyobyo Kenkyu 26(3), 109–117. Westen, H. & Bainton, D. F. (1979). Association of alkaline phosphatase positive reticulum cells in bone marrow with granulocytic precursors. Journal of Experimental Medicine 150, 919–937. Whitlock, C. A. & Witte, O. N. (1987). Long-term culture of murine bone marrow precursors of B lymphocytes. Methods in Enzymology 150, 275–288. Wolf, K. & Quimby, M. C. (1969). Fish cell and tissue culture. In Fish Physiology (W. S. Hoar & D. S. Randall, eds) pp. 253–305. New York: Academic Press. Worton, R. G. & Du#, C. (1979). Karyotyping. In Methods in Enzymology (W. B. Jakoby, ed.) 58, pp. 322–344. London: Academic Press. Yoshida, H. & Yumoto, T. (1987). Alkaline phosphatase-positive reticular cells of chicken bone marrow in vivo and in vitro studies. International Journal of Cell Cloning 5, 35–54. Zapata, A. (1979). Ultrastructural study of the teleost fish kidney. Developmental and Comparative Immunology 3, 55–65. Zapata, A. (1983). Phylogeny of the fish immune system. Bulletin de l’Institut Pasteur 81, 165–186.
Establishment and characterization of a pronephric stromal cell line (TPS) from rainbow trout, Oncorhynchus m ykiss W. M. L. DIAGO, M. P. LÓPEZ-FIERRO, B. RAZQUIN
AND
A. VILLENA*
Departamento de Biología Celular y Anatomía, Facultad de Biología, Universidad de León, 24071 León, Spain (Received 22 February 1995, accepted in revised form 3 May 1995) A new stromal adherent cell line, called trout pronephric stroma (TPS), was initiated from a long-term pronephric culture of an adult rainbow trout, and subcultured 104 times over a period of 4 years. This study describes the culture conditions and characterization by enzyme-cytochemistry, electron microscopy, isoenzyme profile, cytogenetic techniques, and viral susceptibility. The cell types in TPS cultures consisted of fibroblastic, epithelioid and giant cells. The modal chromosome number is 58. The optimum growth temperature of the TPS cells is 18–22) C, with a population doubling time of 27 h. The TPS line is susceptible to viral haemorrhagic septicaemia (VHS) virus and infectious haemopoietic necrosis (IHN) virus. ? 1995 Academic Press Limited Key words:
cell culture, cell line, pronephros, stromal cells, rainbow trout, fish.
I. Introduction The pronephros of teleosts is an important haemopoietic organ, in which all lines of haemopoietic di#erentiation have been observed, including a considerable capacity for lymphopoiesis (Smith et al., 1970; Zapata, 1979, 1983). Moreover, it has been proposed as a primary lymphoid organ for the production of B cells in salmonids (Kaatari & Irwin, 1985). From ultrastructural (Zapata, 1979) and histochemical (Castillo et al., 1987; Castillo, 1991) studies, the pronephros of O. mykiss has been shown to contain reticular and sinusoidal endothelial cells, the characteristics of which are similar to those of their counterparts in the bone marrow of mammals (Westen & Bainton, 1979) and birds (Yoshida & Yumoto, 1987). Therefore, the trout pronephros would appear to contain haemopoietic microenvironments resembling those of the bone marrow of the higher vertebrates. The techniques of long-term bone marrow cultures (Dexter et al., 1977; Whitlock & Witte, 1987) have provided an excellent methodology for studying the role of haemopoietic microenvironments in mammals (Allen, 1981; Dexter et al., 1990). We have applied these techniques to established long-term cultures of rainbow trout pronephros capable of supporting haemopoiesis (Diago et al., 1993a). From one of such long-term cultures, we have obtained a *Author to whom correspondence should be addressed. 441 1050–4648/95/060441+17 $12.00/0
? 1995 Academic Press Limited
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new stromal adherent cell line, which we call trout pronephric stroma (TPS). The TPS cell line is able to support the development of haemopoiesis when it is co-cultured with haemopoietic precursors (Diago et al., 1993b). In this paper we describe the culture conditions, phenotypical characterization, growth characteristics, isoenzyme profile and viral susceptibility of the TPS cell line.
II. Materials and Methods CULTURE CONDITIONS
The cell line was derived from a healthy adult rainbow trout, Oncorhynchus mykiss (Walbaum), obtained from a commercial fish farm. After anaesthesia with tricaine methane sulphonate (Sandoz), blood was extracted from the caudal sinus and the pronephros was dissected in sterile conditions and placed in 0.16 M phosphate-bu#ered saline (PBS) Ca2+ and Mg2+ free. Long-term cultures were maintained for 8 months (five passages) under the culture conditions described by Diago et al. (1993a). From that time onwards, the TPS cell line was subcultured weekly by trypsinization with 0·05% trypsin–0·02% EDTA solution (Boehringer Mannheim) in PBS, for 3 min at 18&1) C, and 3·5–5#105 cells were transferred to 25 cm2 flasks (Costar) and incubated at 18&1) C in air. At di#erent passages the TPS cell line was cryopreserved, using 1·5 ml Nunc cryovials containing 1–3#106 cells per ml in FCS with 10% dimethylsulphoxide (DMSO). The cells were stored in liquid nitrogen, and cultures were routinely re-established from frozen stocks. The culture medium was RPMI-1640 (BioWhittacker) with L-glutamine and 25 mM Hepes bu#er supplemented with 2 mM L-glutamine (Biochrom), 2·5 mM sodium pyruvate (Merck), 50 ìg ml "1 gentamycin (Biochrom), nucleosides (Sigma)—0·088 mM guanosine, 0·225 mM cytidine, 0·094 mM adenosine, 0·102 mM uridine—, 5#10 "5 M 2-mercaptoethanol (Merck), 10% FCS (Boehringer Mannheim) and 5% allogenic trout serum. After the adjustment of the pH to 7·4–7·6, and the osmolarity to 295·33 mOs kg "1 of water, the medium was sterilized by filtration.
ELECTRON MICROSCOPY
At passage 58 the TPS cells were fixed in situ with cold 2·5% glutaraldehyde in cacodylate bu#er (0·2 M, pH 7·2) for 2 h at 4) C, and post-fixed in 1% osmium tetroxide in the same bu#er for 1 h at 4) C. After two rinses with cold distilled water, cells were scraped o#, pelleted and given consistency using 2% agar. The pieces were then dehydrated in acetone, contrasted with 1% uranyl acetate in 70% acetone and embedded in araldite (Durcupan ACM). Semithin and ultrathin sections were obtained with an Ultracut-E ultratome (Reichert-Jung). The semithin sections were stained with a 1% aqueous solution of toluidine blue in borax to select appropriate areas. Ultrathin sections were counterstained with lead citrate (Reynolds, 1963) and examined with a JEOL EM-1010 electron microscope at 60 Kv.
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Table 1. Monoclonal (m) and polyclonal (p) antibodies used in phenotyping the TPS cell line Primary antibodies Anti-Keratin (p) Anti-S100 (p) Anti-Collagen I (p) Anti-Collagen III (p) Anti-Vimentin Clon VIM3B4 (m)
Specificity
Source
Bovine pan-Keratin Bovine S100 protein Human & Bovine Collagen I Human & Bovine Collagen III Bovine Vimentin
Dakopatts Sigma Southern. Biotech. Assoc.
Mouse Ig Rabbit IgG Goat Ig
Dakopatts Sigma Dakopatts
Southern. Biotech. Assoc. Boehringer Mannheim
Secondary antibodies Rabbit anti-mouse Ig Goat anti-rabbit Ig Rabbit anti-goat Ig
CYTOCHEMICAL ANALYSIS
At passages 14, 34, 60 and 82, the following enzyme activities were assayed: acid phosphatase (ACPH) (Barka & Anderson, 1962), alkaline phosphatase (AKPH) (Burstone, 1958) and non-specific alpha-naphthyl acetate esterase (ANAE) (Pearse, 1972). Negative controls for the specificity of the enzyme reactions were established using incubation media in which the corresponding substrates were lacking. IMMUNOCYTOCHEMICAL ANALYSIS
At the same passages indicated above, primary antibodies against markers of specific mammalian cell types and of connective tissue fibres, indicated in Table 1, were used to characterize the TPS cell line by means of the indirect immunoperoxidase method. Endogenous peroxidase was blocked with 0·3% H2O2 in methanol for 10 min before incubation with the primary antibodies. TPS cultures were incubated for 1 h with appropriate dilutions of the monoclonal or polyclonal antibodies, and then incubated for 30 min with appropriate solutions of peroxidase-conjugated antibodies in 0·16 M PBS (Table 1). The peroxidase reaction was developed with 3,3-diaminobenzidine tetrahydrochloride (Sigma) 0·5 mg ml "1 in Tris-HCl 0·05 M, pH 7·6, plus 0·03% H2O2. Positive controls were carried out on cryostat tissue sections from the mammalian species against which the primary antibodies were produced, and from trout kidney, spleen, thymus, skin and gut. Negative controls for specific immunostaining of the TPS cell line, as well as trout and mammalian tissues, were performed by omitting the primary antibody. PHAGOCYTIC ASSAY
At passage 34, a phagocytic assay was performed by incubating TPS cultures for 1 h at 18&1) C with heat-inactivated yeast (Candida lipolytica)
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stained with neutral red (0·5% in 96% ethanol). A total of 200 cells per flask were counted, and the percentage of cells with yeasts engulfed in their cytoplasm was then calculated.
CYTOGENETIC TECHNIQUES
Karyotype Karyotype analyses of the TPS cell line (at passages 6, 16, 28, 42, 60, 62, 74) were performed as described by Worton & Du# (1979). The chromosomes were stained with a 4% Giemsa solution for 10–15 min, and spreads of 100 cells were counted. NOR staining Nucleolar organizer regions (NORs) were silver-stained by Gold & Ellison’s method (1982).
ISOZYME ANALYSIS
Isozyme profiles at passage 74 were generated using horizontal starch gel electrophoresis and the specific enzymes assayed as described by Taggart et al. (1981). Cells were homogenized in an equal volume of PTP [0·05 M Pipes (Sigma), 0·05% Triton X-100 (Sigma), 0·2 mM Pyridoxal-5*-phosphate (Sigma), pH 6·8]. Gels were assayed for the following enzymes: adenylate kinase (AK), alcohol dehydrogenase (ADH), aspartate aminotransferase (AAT), creatine kinase (CK), fructose 1, 6 diphosphatase (FDP), fumarase (FUM), glucose-6phosphate dehydrogenase (G-6-PDH), glycerol-3-phosphate dehydrogenase (G-3-PDH), hexokinase (HK), isocitrate dehydrogenase (IDH), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), malic enzyme (ME), phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (6-PGDH), phosphoglucose isomerase (PGI), phosphomannose isomerase (PMI), sorbitol dehydrogenase (SDH). Isozyme profiles of the TPS cell line were compared with those from the following cell lines: RTG-2 (from gonad tissue of rainbow trout), CHSE-214 (from Chinook salmon embryo), and BF-2 (from bluegill fry caudal trunk).
CELL GROWTH
Rate of growth The growth of the TPS cell line (at passage 65) was analysed in triplicate as described by Freshney (1987). Briefly, 105, 3#104 and 104 cells per well in 1 ml were seeded into 24-well plates (Costar). In addition, 1·6#104, 4·8#103 and 1·6#103 cells per well in 200 ìl were seeded into 96-well plates (flat-bottom) (Costar). In all cases, cells were incubated at 18&1) C. Cells from three wells (24-well plates), and from 12 wells (96-well plates) were daily removed using trypsin/EDTA and the cell number counted using a haemocytometer. Cell viability was calculated by the trypan blue exclusion method.
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Optimum growth temperature To determine the optimum growth temperature 24-well plates were seeded with 3#104 cells per well in 1 ml, and incubated at 4, 10, 12, 18 and 22) C. The experiment was done in triplicate and extended over 15 days. Cells were harvested daily, counted, and the cell viability determined as described above. 3
H-thymidine incorporation
Rates of DNA synthesis by incorporation of 3H-thymidine were determined in duplicate in 96-well plates (flat-bottom) over 20 days. TPS cells (at passage 65) were plated at 1·6#104, 4·8#103 and 1·6#103 cells per well in 200 ìl and incubated at 18&1) C. Cultures were pulsed with 10 ìl of a 50 ìCi ml "1 3 H-thymidine solution (DuPont) that was added to each culture well. Cells were harvested 24 h later by water lysis onto glass-fibre filters using a semi-automatic cell harvester (Skatron, Norway). Filter discs were dried for 30 min at 80) C, placed into scintillation vials, and 5 ml of scintillation cocktail (Biogreen-1, Scharlau) was added per vial. The number of disintegrations per minute (DPM) were calculated using a calibrated scintillation counter (Beckman LS 6000 TA). SUSCEPTIBILITY TO VIRUSES
Confluent cultures in 25 cm2 flasks were infected with the fish viruses causing infectious haemopoietic necrosis (IHN-V Cedar strain), infectious pancreatic necrosis (IPN-V strain 299), and viral haemorrhagic septicaemia (VHS-V strain 0.7.71), at multiplicities of infection (Basurco & Coll, 1989) ranging from 0·001 to 0·5. The infections were performed as previously described (Diago et al., 1993), and the infected cultures were incubated at 14) C. They were observed with an inverted microscope (Nikon) to determine the occurrence of a cytopathic e#ect. Parallel control uninfected cultures were maintained in the same conditions. III. Results From the earlier subcultures, the TPS cell line comprised adherent cells, mainly showing fibroblastic morphologies. These cells did not show the contact inhibition property, as they did not form true monolayers, some growing over others, even before reaching confluence in the flask. After 104 passages, the TPS cell line has maintained these characteristics. MORPHOLOGY OF THE ESTABLISHED TPS CELL LINE
By light microscopy, initially after a passage, the TPS cells grew forming cell clusters, in which large cells, with multilobulated nuclei, appeared surrounded by fibroblastic cells [Fig. 1(a)]. Later on, TPS cultures did not form true monolayers, but the cells overlapped each other in a multilayered network [Fig. 1(b)]. Confluent TPS cultures consisted of three morphologically di#erent adherent cell types: (a) Elongated or fusiform fibroblastic cells, with
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Fig. 1. Phase contrast micrograph of TPS cultures. (a) 3-day-old culture containing a giant cell (G) and fibroblastic cells (]), #199; (b) general aspect of a 1-week-old culture formed by overlapping fibroblastic (]) and epithelioid (x) cells. Note the giant cell (G), #274; (c) detail of a 1-week-old culture showing the characteristics of the epithelioid cells (x), #364; (d) detail of a 1-week-old culture in which a giant cell (G) is partially overlapped by fibroblastic cells (]), G indicates cell in mitosis, #366.
ovoid nuclei and filiform processes [Fig. 1(b)] formed the major component of the culture; (b) Epithelioid cells, with flattened prismatic cell bodies, which sometimes contained numerous small vesicles, and round nuclei [Figs 1(b) & (c)]; and (c) A low number of giant cells [Figs 1(b) & (d)], resembling those found in the early cell clusters [Fig. 1(a)], with very large rounded cell bodies. Cells of this type showed apparently multilobulated nuclei [Fig. 1(a)] or were multinucleated, most of their organelles, which included clear vesicles, appearing clustered around the nuclear area [Fig. 1(d)]. Ultrastructurally, the cultures consisted mainly of several overlapping layers of elongated cells, with intercellular spaces partially filled by filamentous material [Fig. 2(a)]. The fibroblastic cells had tapered ends and large
Fig. 2. Ultrastructure of the TPS cell line. (a) Multilayered fibroblastic cells. Nucleus (N), rough endoplasmic reticulum (]), multivesicular body (c), note the filamentous extracellular material (*) and the cell-cell contacts ( ), #12 000; (b) higher magnification of the cytoplasm of fibroblastic cells showing elongated (]) and dilated cisternae (G) of rough endoplasmic reticulum, and microfilaments (c), note the extracellular matrix (*), nucleus (N). #30 000; (c) micrograph showing two epithelioid cells (E), nucleus (N), Golgi apparatus (]), and the characteristic vesicles (G), #8000; (d) higher magnification of the cytoplasm of an epithelioid cell showing microfilaments (c) and a desmosome ( ), #25 000.
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Fig. 3. Ultrastructure of the TPS cell line. (a) Micrograph showing part of a giant cell (G) and two epithelioid cells (E). Note two nuclei (N1-N2) of the giant cell. Golgi apparatus (]), #10 000; (b) high-power magnification of the fibrillar material present in the extracellular spaces, fibroblastic cell (F), #40 000.
slender cell branches. They exhibited fusiform nuclei with a band of perinuclear heterochromatin, and one or more nucleoli [Fig. 2(a)]. Major cytoplasmic organelles were numerous free ribosomes, and a well developed rough endoplasmic reticulum, formed by elongated and dilated cisternae containing a material of moderate electron density [Fig. 2(b)]. Some mitochondria, dictiosomes of the Golgi apparatus, short bundles of microfilaments, multivesicular bodies, and some dense bodies were also observed [Figs 2(a) & (b)]. Although there were close appositions of the cell membranes of neighbouring fibroblastic cells [Fig. 2(a)], no cell junctions were observed between them, or with other cell types. Epithelioid cells were characterized by their lobulated, irregular, euchromatic nuclei, and large cytoplasms [Fig. 2(c)], which contained a very well-developed Golgi apparatus, some rough and smooth endoplasmic reticulum, and numerous mitochondria of high electron density [Figs 2(c) & 3(a)]. The presence of numerous, large, electron-clear vesicles, sometimes containing heterogeneous material, was a characteristic marker of the epithelioid cells [Figs 2(c) & 3(a)]. They also contained microfilaments, and were occasionally joined to one another by desmosome junctions [Fig. 2(d)]. Giant cells were very di$cult to recognize in the ultrathin cross-sections, due to the fact that they extend over long, but narrow, areas of the
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Table 2. Cytochemical patterns of the TPS cell line (passage 60 and 82; the latter is in brackets) AKPH
ACPH
ANAE
"(") "(") "(")
"(") "(+/") "(+/")
"(+/") "(+/") + +/+(+ +/+)
Fibroblastic cells Epithelioid cells Giant cells
multilayers. They showed ultrastructural features similar to those described for the epithelioid cells, but with more lobulated and irregular nuclei, and sometimes appearing multinucleated [Fig. 3(a)]. The intercellular substance between the cells [Fig. 2(a) & (b)] was formed by amorphous materials and some striated fibrils [Fig. 3(b)]. CYTOCHEMICAL ANALYSIS
Controls for the enzyme activities assayed were always negative. The cytochemical features of the cell types found in the TPS cultures (passages 60 and 82) are summarized in Table 2. IMMUNOCYTOCHEMICAL ANALYSIS
Positive controls confirmed that the primary antibodies to mammalian markers cross-reacted with components in the trout tissues. However, TPS cells did not stain with antibodies to mammalian keratin, collagen types I and III, vimentin and S-100 protein. PHAGOCYTIC ASSAY
After 1 h of co-culture of TPS cell line and C. lipolytica, only 1% of the cells had engulfed yeasts. CYTOGENETIC TECHNIQUES
Chromosome counts Chromosome analyses showed the TPS cells to be heteroploid. They contained from 46 to 66 chromosomes, more metacentric (42–50) than acrocentric (10–18), which showed a gradual variation in size. The modal chromosome number of the TPS cell line found at passage 60 was 58. Frequency distribution of chromosomes in 100 cells is shown in Fig. 4. Ag-NOR staining Two NOR-bearing chromosomes were seen in all silver-stained metaphases (Fig. 5). This chromosome pair was submetacentric, with the NOR located in the long arms of the two chromosomes.
M. L. DIAGO ET AL.
450 30
25
Cell number
20
15
10
5
0 44 46 48 50 52 54 56 58 60 62 64 66 68 Chromosome number
Fig. 4. Frequency distribution of chromosomes in TPS cells per 100 cells.
Fig. 5. Metaphase chromosomes of the TPS cell line stained for demonstration of NOR-bearing chromosomes (]), #1719.
ISOZYME ANALYSIS
The TPS cell line was positive for the isozymes shown in Table 3. In addition, in these assays the relative electrophoretic migration of each isozyme was used to di#erentiate the TPS cell line from other cell lines (Table 3). Thus, the four cell lines could be di#erentiated by the isozyme profiles for the malate dehydrogenase (MDH). Moreover, the TPS cell line could be
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Table 3. Comparison of the isozyme profiles between the TPS cell line and other fish cell lines Isozyme
TPS v. BF-2
TPS v. RTG-2
TPS v. CHSE-214
AK CK FDP HK IDH LDH MDH 6-PGDH PGI PGM PMI
1 1 3 1 1 1 1 1 2 1 2
2 1 3 2 1 2 1 2 2 1 3
2 3 2 3 3 2 1 2 2 3 3
1, di#erent profile; 2, identical profile; 3, detectable activity in the TPS cell line but no activity in the other cell lines.
di#erentiated from the BF-2 cell line by the AK, CK, HK, IDH, LDH, 6-PGDH, and PGM isozymes and from the RTG-2 cell line by the isozyme profiles for the CK, IDH and PGM. The other seven enzymes assayed were not suitable for di#erentiating these fish cell lines, because they lacked detectable activity in the majority of the lines or had blurred profiles.
CELL GROWTH
TPS cells are relatively easy to maintain as adherent cultures in plastic flasks or dishes. Attempts to grow TPS cell line in suspension cultures have been unsuccessful. Rate of growth The growth curves of the TPS cell line (passage 65) at 18) C, in 24-well and 96-well plates are represented in Figs 6(a) & (b) respectively. The population doubling time, calculated from the middle of the exponential phase of growth for the initial cellular density of 3#104 cells ml "1 (1·5#104 cells per cm2), was 27 h. Optimum growth temperature Growth curves for the TPS cell line cultured at di#erent temperatures are shown in Fig. 6(c). At 10) C or 12) C no increase in cell numbers was observed for 15 days. In the range of temperature tested, optimum growth was at 18–22) C. At 4) C the cells remained viable and cell growth continued very slowly over long periods of time. In this case, TPS cells maintained in 25 cm2 flasks could be subcultured once a year.
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452 (a)
(b) 5
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104 0 2 4 6 8 10 12 14 16 18 20 22
0 2 4 6 8 10 12 14 16 18 20 22
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8 10 12 14 16 Days
Fig. 6. Growth curves of TPS cells. (a) In 24-well plates (4) 105 c/w, (/) 3#104 c/w, (-) 104 c/w; (b) in 96-well plates (4) 1·6#104 c/w, (/) 4·8#103 c/w, (-) 1·6#103 c/w; (c) at various temperatures (;) 22) C, (4) 18) C, (/) 12) C, (-) 10) C. c/w (cells per well) indicates starting seeding densities. Each point represents the mean of three counts& S.D. 3
H-thymidine incorporation At a seeding density of 1·6#104 cells per well (5#104 cells per cm2) maximal 3 H-thymidine incorporation was reached after 6 days in culture (Fig. 7). This peak corresponds with the increase in the cell number which was observed between days 6 and 8 of culture [Fig. 6(b)]. At a seeding density of 4·8#103 cells per well (1·5#104 cells per cm2), maximum 3H-thymidine incorporation was reached after 12 culture days. At a seeding density of 1·6#103 cells per well (5#103 cells per cm2) there was a continuous increase in the incorporation of 3H-thymidine for the duration of the experiment. SUSCEPTIBILITY TO VIRUSES
VHS and IHN viruses, produced a cytopathic e#ect (CPE) but IPN-V did not. The CPE began with detachment of scattered cells after 3–5 days. At 7 days post-infection or later depending on the multiplicity of infection, infected cultures showed small areas devoid of cells. Cultures were totally lysed by three weeks post-infection with VHS-V and IHN-V regardless of the initial multiplicity of infection used.
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DPM
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Fig. 7. Kinetics of 3H-thymidine incorporation in TPS cells grown at 18) C. c/w (cells per well) indicates starting seeding densities. Each point represents the mean of two rows (n=24)& S.D. (4) 1·6#104 c/w, (/) 4·8#103 c/w, (-) 1·6#103 c/w.
IV. Discussion Although there are in vitro studies concerning the immunological capacity of the fish pronephros, and some kidney stromal cell lines have been established (Chen & Kou, 1987; Tung et al., 1991), no special attention has been paid to the development of long-term pronephric cultures, which may support in vitro haemopoiesis. Recently, long-term cultures from the pronephros of O. mykiss, which are able to support in vitro haemopoiesis have been developed (Diago et al., 1993a; Siegl et al., 1993). From one of such long-term pronephric cultures started in 1990, we have established the TPS cell line described here. The successive subcultures eventually lost the haemopoietic capacity, and became formed only of stromal cell types. The TPS cell line is comprised of fibroblastic, epithelioid and giant cell populations, distinguishable by their morphology and cytoenzyme profiles. These cell types grow in culture forming multilayered networks, which resemble those found in the long-term haemopoietic cultures of the pronephros (Diago et al., 1993a; Siegl et al., 1993). This kind of arrangement is also similar to that observed in long-term cultures of mammalian bone marrow, and the zones where cultured stromal cells arrange themselves tridimensionally are likely to be sites of haemopoiesis (Spooncer & Dexter, 1984). This features of the TPS cell line could explain its ability to support the development of haemopoiesis when it is co-cultured with haemopoietic precursors (Diago et al., 1993b). Stromal cell types similar to the fibroblastic and to the epithelioid cells observed in the TPS cell line are also present in the long-term haemopoietic cultures of the pronephros (Diago et al., 1993a; Siegl et al., 1993). The fibroblastic cells resemble the reticular cells which form the stroma of the
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pronephros in vivo. However, the fibroblastic cells of the TPS cell line lack alkaline phosphatase activity, an enzyme marker of reticular cells of the trout pronephros (Castillo et al., 1987), and also of the mammalian (Westen & Bainton, 1979; Bainton et al., 1986) and bird (Yoshida & Yumoto, 1987) haemopoietic bone marrow. This activity has been associated with the secretion by these cells of either elements of the tissue matrix (ground substance and connective fibres) or haemopoietic factors (Landreth et al., 1981; Song et al., 1985; Dorshkind, 1987). In this respect, there occurred masses of amorphous and fibrillar materials in the intercellular spaces of the cultures. The morphology of the fibrillar material resembles that of the reticulin fibres, thus suggesting that the TPS cell line is able to release components of the connective tissue matrix. This was also true of the long-term haemopoietic cultures of the pronephros (Diago et al., 1993a). However, immunostaining for collagen types I or III in the TPS cultures was negative, which may indicate that the collagen-like fibres are not of these collagen types or, alternatively, that there occurred a defective collagen synthesis in vitro, which resulted in the loss of its antigenic properties. The epithelioid and giant cells show similar structural features and cytoenzyme profiles. From our observations, we think that giant cells may di#erentiate from epithelioid cells. We also observed epithelioid cells in long-term pronephric cultures (Diago et al., 1993a), and we related them to the sinusoidal endothelial cells. The presence of multinucleate giant cells has been described in long-term pronephric cultures (Siegl et al., 1993), and this fact has been related to the ability of macrophages to fuse in culture (Secombes, 1985). Nevertheless, we have not observed fusion of epithelioid cells in the TPS cultures. Although the epithelioid and giant cells show ANAE activity, an enzyme marker of trout macrophages (Razquin et al., 1990), there are no melanomacrophages or typical macrophages in the TPS cell line. Nor is there any remarkable phagocytic activity. Moreover, the epithelioid and giant cell types were always negative for all the cell immuno-markers we have tested, and so their cell lineage remains obscure. The TPS cell line seems to remain cytogenetically stable through the passages, as demonstrated by classical karyotyping. The type and modal number of chromosomes of the cell line are in the range of the values described for rainbow trout (Thorgaard, 1976; Hartley, 1987). Moreover, our results from the selective staining of the NORs agree with those reported for the normal diploid karyotype of rainbow trout (Phillips & Ihssen, 1985; Lozano et al., 1992). Isoenzyme analysis demonstrated that the malate dehydrogenase (MDH) isozyme profile is useful to distinguish the fish cell lines studied here. The profiles for IDH isoenzyme also demonstrated di#erences between the TPS and the RTG-2 cell lines. Growth at various incubation temperatures was evaluated in several experiments to determine the range of growth temperatures as well as the optimum growth temperature for TPS cells. Like other salmonid cell lines (Gravell & Malsberger, 1965; Wolf & Quimby, 1969; Bruce & Byrne, 1973), TPS cells are capable of growth throughout a temperature range from 4) C to 22) C, with maximum growth at 18) C to 22) C. Determination of rate of DNA synthesis by
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incorporation of 3H-thymidine demonstrated that a detectable activity remained for the duration of the experiment. This residual incorporation rate is probably due to (a) the ability of the cells to grow in multilayers, and (b) to the replacement of dead cells, and confirms that there is no contact inhibition in the TPS cell line. The susceptibility of the TPS cell line to IHN-V and to VHS-V agrees with that found for the pronephric stromal cells to VHS-V (Diago et al., 1993), thus indicating that this property has been retained throughout the successive passages. This feature would allow the use of the TPS cell line to analyse the pathogenic mechanisms of these viral agents, as well as for diagnostic purposes. On the other hand, haemopoiesis is regulated by a delicate balance between cell-cell interactions and a network of numerous growth factors, some of them released by blood cells, and others by stromal cells (Dorshkind, 1990). This network of cytokines and growth factors is probably also present in fish (Graham & Secombes, 1990), and cell-cell contacts between developing cells and the stromal cells appear to be necessary for trout haemopoiesis (Diago et al., 1993a). The TPS cell line, which seems to maintain the cellular components and spatial relationships found in the main haemopoietic tissue of trout, may represent a useful tool for studying the mechanisms regulating fish haemopoiesis. This work has been partially funded by the Spanish CICYT, project No. MAR-910851, and the III Framework Programme of the EU, Contract AIR1 CT92 0036. The authors thank Dr P. Martínez (Universidad de Santiago, Lugo, Spain) for the technical assistance with the isozyme analyses. Viral infections were done at the high biological security laboratory (P3) of the INIA-CISA (Madrid, Spain), under the supervision of Dr J. Coll. We also thank the ‘Piscifactoría Los Leoneses’ for providing rainbow trout. M. L. Diago holds a fellowship from the Diputación Provincial de León.
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