- Email: [email protected]
Primary culture of rainbow trout corpuscles of Stannius
Hochachka
and Mommsen
(eds.), Biochemistry
and molecular
biology of fishes, vol. 3
© 1994 Elsevier Science B.V. All rights reserved. C H A P T E R 23
Primary culture of rainbow trout corpuscles of Stannius BIRGIT GELLERSEN AND GRAHAM F. W A G N E R * Institute for Hormone and Fertility Research, Grandweg 64, 2000 Hamburg 54, Germany, and * Department of Physiology, Faculty of Medicine, University of Western Ontario, London, Ontario, Canada N6A 5C1
I. II.
Introduction Preparation of cell cultures 1. Harvesting of corpuscles of Stannius 2. Cell preparation III. Comments 1. Cell preparation 2. Choice of media 3. Culture conditions 3.1. Temperature 3.2. Osmotic pressure 3.3. Calcium content 4. Handling and storage of conditioned media IV. R N A extraction from plated CS cells Acknowledgements V. References
/.
Introduction
The corpuscles of Stannius (CS) are the source of stanniocalcin (STC), a hormone that is secreted in response to hypercalcemia and acts to inhibit calcium transport across the fish gill. These glands are ideally suited for primary culture, as they are readily accessible in most species and can be obtained in quantity to yield large numbers of cells. In this chapter, we have provided a detailed procedure for preparing and maintaining primary cultured rainbow trout (Oncorhynchus mykiss) CS cells. For those who are so inclined, we offer encouragement by stressing the ease with which these cells can be maintained in culture, with a minimum amount of expense, equipment and practice. We have also included media recipes for two different styles of culture, one requiring gas ( C O 2 ) and another that does not. Both techniques work well as we have used them both. The first technique employs commercially available D M E M or amphibian media, and requires a minimal amount of gassing and a cold cabinet kept at 10-15°C. This
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is the method we used to set up our first primary cultures of trout CS cells . The advantage of this method is that it doesn't require an incubator and is therefore a low-cost approach. The main disadvantage of the method is that the cultures have to be monitored daily to ensure pH stability. It also requires a cold cabinet or similar environment maintained at a constant, low temperature (10-15°C). Therefore, either a refrigerator or a cold room would have to be dedicated exclusively to culturing CS cells. Alternatively, this method can be adapted directly for use with a C O 2 incubator. The second technique employs Leibovitz media (L-15), makes use of an incuba tor maintained at 10-15°C in a 4°C cold room and does not require gas. Hence, the incubator is used only for maintaining sterility and a stable, low temperature. The disadvantage of this method is that it requires an incubator and therefore entails extra costs. However, if an incubator can be obtained at little or no expense (an old C O 2 incubator will suffice), then this is the method of choice. The cultures require little attendance except for occasional feeding, pH stability is not an issue and most if not all institutions have a 4°C cold room. This is the method we now employ with excellent results.
77. Preparation of cell cultures 1. Harvesting of corpuscles of Stannius (day 1) Have the following materials at hand: (a) Sterile dissecting instruments (fine forceps, scissors, scalpels, alcohol lamp, 70% ethanol) (b) 50 ml tissue culture tubes with ca. 20 ml transport media on ice Kill fish with a blow to the head, slit the gills and drain the blood from the carcass by holding the animal by the tail in a vertical position. Then cut open the abdomen in a forward direction from the vent and remove the viscera and swim bladder. Usually there are three or four CS (small cream-coloured glands) in trout, midway along the kidney attached to the ventral margins or buried in and protruding from the dorsal surface of the kidney. Pierce the thin sheath of connective tissue covering the kidneys with fine forceps, remove the CS and place them in ice-cold transport media. Rinse forceps in 70% ethanol and pass them over a flame after each dissection. If glands are required from a large number of fish, an alternative procedure used at some Canadian fish hatcheries and modified for our own use is more expedient. A 1 χ 0.4 meter sheet of wood or plexiglass with 6-10 nails protruding from the front surface (along the top) is secured in a near-verticle position, preferably over a sink basin. After stunning each fish, impail them by the tail on the nails such that they hang vertically, head downward. With the fish secured in this manner, the bleeding, evisceration and dissection procedures can be done by one person. We routinely use this procedure to obtain glands from 40-50 fish in 2 - 3 hours. Keep the glands in transport media on ice until returning to the laboratory.
Primary culture of rainbow trout corpuscles of Stannius
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2. Cell preparation Cell (a) (b) (c) (d) (e) (f) (g) (h) (i) (j)
preparation (day 1). Have the following materials at hand in the laboratory: sterile forceps, scalpels and 21 gauge needles, sterile glass petri dishes on ice, sterile Pasteur pipets (e.g. plastic liquipettes), 50 ml conical tissue culture centrifuge tubes, bench top centrifuge, sterile pipets and pipet tips, verticle turntable or shaker in a cold room, hemocytometer, 2 tissue culture vessels such as 24-well plates, 25-cm flasks, etc., a dissecting microscope.
All procedures involved in preparing the cell suspension are done in a sterile, laminar flow hood, keeping the tissue and solutions at 4°C whenever possible: Step 1. Decant glands from transport tube into chilled petri dish, rinse twice with transport media by aspirating wasted media with a pasteur pipet. Step 2. To drained CS, add a few ml of dissecting media and remove residual renal and fat tissue with fine forceps. Step 3. Transfer clean glands to a new petri dish containing a few ml of dissecting media. Step 4. Tear glands into small pieces with fine forceps. Step 5. Transfer suspension to 50-ml tube, spin 8 min at 1000 rpm (250 g). Step 6. Save pellet, transfer supernatant to new 50-ml tubes, sediment as above, discard supernatant, save second pellet. Step 7. Add 10 ml of trypsin solution to each of the two tubes. Step 8. Close tubes tightly and attach to a slow moving turntable in the cold room for overnight (16 h) digestion at 4-8°C (alternatively, place tubes in a horizontal position in a styrofoam box filled with ice and agitate on a shaker). Cell preparation (day 2). Step 1. Add 50 μΐ DNase (5 /xg/ml final cone.) per 10 ml of trypsinized cells. Step 2. Agitate 15 min at room temperature in a horizontal position. Step 3. Decant suspension into petri dish, tease apart undigested tissue with nee dles or fine forceps to aid dissociation of cells, remove undigested tissue fragments. Step 4. To break up cell clumps draw the cell suspension repeatedly through a 10 ml pipette, all the time pressing the pipette tip against the bottom of the petri dish. Step 5. Filter cell suspension with a Falcon cell strainer into a 50 ml centrifuge tube, centrifuge cells for 8 min at 1000 rpm. Step 6. Save pellet, transfer supernatant to new tube, sediment again as above, discard supernatant. Step 7. Wash both pellets with 10 ml of dissection media, centrifuge 8 min at 1000 g, discard supernatant. Step 8. Pool pellets after resuspension of cells in culture media (use 10 ml media for CS obtained from 10 fish).
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TABLE 1 Stock media formulations for gassed cultures (to make 1000 ml each) RPMI-1640 Stock with reduced NaHC03 - dissolve powder (without N a H C 0 3 , with L-Glutamine-Gibco) in 900 ml H 2 O - add 0.76 g N a H C 0 3 (to give final cone, of 9 mM) - adjust pH to 7.2 with 1 Ν HCl - make up to 1 liter, filter sterilize, keep at 4°C DM EM -
Stock (Dulbecco's Modified Eagle Medium with reduced NaHCOj dissolve powder (without N a H C 0 3 , with L-Glutamine-Gibco) in 900 ml H 2 O add 2.45 g N a H C 0 3 (to give final cone, of 30 mM) adjust pH to 7.2 with 1 Ν HCl make up to 1 liter, filter sterilize, keep at 4°C
MEM Stock (Minimal Essential Medium with Earle's Salts, with L-Glutamine-Gibco) Ready-to-use, keep at 4°C (contains 2.20 g N a H C 0 3 / L , 6.80 g NaCl/L, 0.20 g CaCl 2/L) EBSS Stock (Earle's Buffered Salt Solution) without NaCl 0.20 g C a C l 2 0.40 g KCl 0.20 g M g S 0 4 7 H 2 0 2.20 g N a H C 0 3 0 . 1 4 g N a H 2P O 4H 2O dissolve in 900 ml H 2 0 , adjust pH to 7.2, make up to 1 liter, filter sterilize, keep at 4°C HBSS Stock (Hanks' Balanced Salt Solution, with increased NaHCOj) - dissolve powder (without N a H C 0 3- G i b c o ) , in 900 ml H 2 0 - add 0.76 g N a H C 0 3 (to give 9 mM final cone.) - adjust pH to 7.2 with 1 Ν HCl - make up to 1 liter, filter sterilize, keep at 4°C
Step 9. To determine cell yield and viability, mix 20 μΐ cell suspension and 20 μΐ Trypan blue, wait 10 minutes, count viable cells (unstained) and dead cells (blue), do not count the RBC. 5 Step 10. Dilute cell suspension to 5 χ 10 cells/ml in media of choice and plate 1 2 ml/well in 24-well plates or 8 ml in 25-cm flasks. Gassed cultures. If a C O 2 incubator is unavailable, place cultures in tightly sealed containers. Purge the containers with 5% COil9S% air and maintain them in a cold cabinet (10-15°C). Domestic, plastic containers with water-tight lids are perfectly adequate for maintaining the cultures in a sealed environment. Inlet and outlet ports for purging containers with the CC^/air mixture can be improvised; stopcocked syringe needles can be inserted through the lids and secured in place with adhesive. Supplement gas to the sealed cultures daily, or when the phenol red in the media indicates a pH above 7.5. Chambers which are specifically designed for sealed cultures are commercially available (Caltech Scientific). Alternatively, if a C O 2 incubator is available, place it in a cold room and maintain the cells at 10-15°C, supplemented with a 5% C 0 2 / 9 5 % air mixture. Non-gassed cultures. In the case of cells plated in L-15 media, maintain cultures in a 10-15°C cold cabinet (sealed containers are not required) or an incubator in
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a 4°C cold room, maintained at 10-15°C. Be aware of the fact that cold rooms are notoriously unclean environments. Therefore, clean and rinse all inner surfaces of the incubator with 70% ethanol prior to preparing each new lot of cells. Cell preparation (day 4). Check cultures under an inverted microscope. The cells should have flattened out and attached to the plates, but secretory granules will not be evident at this point. There will also be a lot of cellular debris in the cultures. Aspirate off media with gentle suction, wash cells with HBSS or culture media minus serum and antibiotics, aspirate and replace with fresh media. Cell preparation (day 7). Secretory granules should now be clearly visible in the cells, as illustrated in Panels 3b-6b, and the cultures are ready for experimental manipulation.
III.
Comments
1. Cell preparation The removal of kidney and fat tissue from the glands is best done under a dissecting microscope. The proportion of viable cells after the cold trypsinization should be 6 2 7 5 - 9 5 % with a yield of 1.4-2.4 χ 1 0 viable cells/gland in 2.5 kg trout and 1 6 2 χ 10 cells/fish in 0.5 kg trout (the number and size of individual glands are highly variable between fishes of the same weight). In our experience, it is most convenient to collect the glands on a Tuesday morning (day 1), such that the cells have formed monolayers by Friday (day 4). After washing the cells on day 4, they can be left over the weekend and are ready for experimentation on the following Monday (day 7). 2. Choice of media For gassed cultures, both D M E M and amphibian media work equally well, so choose your media on the basis of cost and availability. RPMI is only required for experiments involving manipulations of media calcium content. D o not prepare or plate cells in RPMI as the calcium content is too low. This may affect cell viability and the cells will take too long to attach to the plates. HEPES buffer is added to stabilize the pH at 7.4-7.5 in the sealed, gassed cultures when a C O 2 incubator is unavailable for use. Nonetheless, occasional gassing will be required to lower the p H and therefore it is best to check the cultures daily. For nongassed cultures, Leibovitz media can be prepared in the laboratory with considerable savings over commercially available media. Feed cells with fresh media at least twice weekly. 3. Culture
conditions
Three variables deserve special interest regarding culture conditions for rainbow trout CS cells: temperature, osmotic pressure and calcium concentration.
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TABLE 2 Media preparation for gassed cultures Prepare fresh from the stock solutions listed in Table 1 before harvesting glands Transport medium 96 ml HBSS stock 2 ml H E P E S (20 mM final cone.) 1 ml Penicillin-Streptomycin (100 U/ml-100 /zg/ml final cone.) 1 ml Fungizone (2.5 μg/ml final cone.) Dissection medium 98 ml transport medium 2 ml B S A (0.2% final cone.) Trypsin solution 18 ml transport medium 0.4 ml B S A (0.2% final cone.) 2 ml Trypsin (0.25% final cone.) Amphibian culture medium 50 ml whole egg ultrafiltrate (10% final cone.) 50 ml FCS (10% final cone.) 125 ml EBSS/NaCl-free stock (25% final cone.) 275 ml MEM stock (55% final cone.) 10 ml H E P E S (20 mM final cone.) 2.5 ml Penicillin-Streptomycin DMEM culture medium 440 ml D M E M stock 50 ml FCS (10% final cone.) 10 ml H E P E S (20 mM final cone.) 2.5 ml Penicillin-Streptomycin RPMI serum-free culture medium (RPMISF) 97 ml RPMI-1640 stock 2 ml H E P E S 0.5 ml Penicillin-Streptomycin 1 ml B S A ( 0 . 1 % final cone.)
*
* Only for experiments requiring low calcium levels.
3.1. Temperature In our view, it is best to culture trout CS cells at 10-15°C to mimic the natural environment. It may also be possible to maintain the cells at room temperature, however we have not tried this. Furthermore, what effects this might have on CS cell metabolism is unknown. Cultures can be maintained "stored" in the fridge without attendance for several weeks. 3.2. Osmotic pressure Rainbow trout inhabit both seawater and fresh water and can therefore adapt to a wide range of salinities. Trout plasma is approximately 300 mOsm, whereas mammalian culture media such as D M E M and RPMI-1640 have values of 2 7 0 275 mOsm, corresponding to mammalian plasma. Nevertheless, trout CS cells do well in these media. The preparation of HBSS buffer with increased NaHCOß, as 5 described in the media formulations, gives a value of 300 mOsm . Interestingly,
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TABLE 3 Stock media and media formulations for nongassed cultures Leibovitz Stock (L-15) with L-glutamine and without NaHCOj (Gibco), - ready to use, keep at 4°C 3 - or prepare according to original recipe (Leibovitz 1963) Transport medium 98 ml L-15 stock 1 ml Penicillin-Streptomycin (100 U/ml-100 ^g/ml final cone.) 1 ml Fungizone (2.5 ^g/ml final cone.) Dissection medium 98 ml transport medium 2 ml B S A (0.2% final cone.) Trypsin solution 18 ml transport medium 0.4 ml B S A (0.2% final cone.) 2 ml Trypsin (0.25% final cone.) Leibovitz culture medium (L-15) 90 ml L-15 stock 10 ml Fetal Bovine Serum 0.5 ml Penicillin-Streptomycin Leibovitz serum-free culture medium (L-15-SF) 99 ml L-15 stock 1 ml B S A 0.5 ml Penicillin-Streptomycin
TABLE 4 Common stock solutions FCS (Fetal Calf Serum): store 50 ml aliquots at - 2 0 ° C * Whole Egg Ultrafiltrate
(Gibco)
BSA (Bovine Serum Albumin Fraction V, cell culture tested) (Sigma). Prepare 10% stock solution, pH 7.4, in HBSS or L-15 medium. Filter sterilize, freeze aliquots Trypsin (Gibco): Ready-to-use 10X stock solution (2.5% in 0.9% NaCl), keep aliquots at - 2 0 ° C Penicillin-Streptomycin (Gibco): Ready-to-use stock solution with 10,000 U Penicillin/ml and 10 mg Streptomycin/ml; keep aliquots at —20°C Fungizone (Gibco): Freeze aliquots of a stock solution containing 250 ^ g Amphotericin B/ml * HEPES (Gibco): Ready-to-use 1 Μ stock solution, keep at 4°C DNase I (Boehringer): Prepare a 1 mg/ml stock solution in phosphate buffered saline, filter sterilize, freeze aliquots Trypan Blue (Boehringer): ready-to-use 0.5% solution in 0.9% NaCl, keep at room temperature * Not required for nongassed cultures
6
Amphibian media which was originally developed for bullfrog cultures is 242 mOsm, but also supports long term maintenance of CS cells. Due to its low osmotic pressure, amphibian media is applicable to experiments designed to test the effects of increasing osmotic pressure.
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Fig. 1. Primary cultured rainbow trout CS cells viewed under brightfield (a) and phase contrast microscopy (b) six days after initial plating in L-15-SF medium (1.2 mM calcium). Panels 1-6 are of CS cells following a one day exposure to L-15 medium containing different concentrations of ionic calcium. Panel la,b. CS cells in 0.3 mM calcium. N o t e the rounded appearance of the cells (arrows in a,b). Panel 2a,b. CS cells in 0.7 mM calcium. N o t e that cells are less rounded (arrows in b). Panel 3a,b. CS cells in 1.1 mM calcium. The cells have flattened and are filled with secretory granules (arrows in b).
3.3. Calcium content For efficient attachment of CS cells after plating, culture media with a "regular" calcium content are recommended. Amphibian and D M E M media when formulated as described, contain 1.67-1.8 mM total C a 2 +, the ionic (free) C a 2 + concentration being somewhat lower due to binding of calcium to serum proteins (Amphibian medium has 1.34 mM ionic C a 2 +) . For experiments designed to test the effects of calcium on STC secretion from CS cells, a media with a low calcium content such as RPMI is desirable. For a limited period of time, CS cultures can be
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Fig. 1 (continued). Panel 4a,b. CS cells in 1.5 mM calcium. The density of cells containing secretory granules (arrows in b) is not noticeably different than cells in 1.1 mM calcium. Panel 5a,b. CS cells in 1.9 mM calcium. Fewer cells contain secretory granules (arrows in b) in comparison to cells in Panels 3 and 4. Panel 6a,b. CS cells in 2.3 mM calcium. Very few cells now contain secretory granules (arrows in b).
maintained in RPMI-SF which has 0.32 mM free C a 2 + (ref. 4). For experiments on the effects of calcium employing L-15 media, we prepare our own stock media using the original formulation, except that calcium content is adjusted to 0.3 mM. In this way, calcium can then be added back to the plated cells to obtain any desired concentration. After initial plating in L-15 media containing normal calcium concentrations (1.2 mM), trout CS cells can then be maintained for at least a week in L-15 containing only 0.3 mM calcium. However, the morphology of the cells changes under these conditions (they round up) and only return to normal (a flat monolayer) when calcium levels are restored (Fig. 1, panels la,b and 3a,b).
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4. Handling and storage of conditioned
Wagner
media
Store conditioned media at - 7 0 ° C and thaw on ice. After thawing, the media should be vortexed and then centrifuged at 12,000 g to pellet cellular debris prior to analysis.
IV RNA extraction from plated CS cells 1
To isolate total R N A from plated cells, use the method of Chomczynski and Sacchi as described. However, after aspiration of conditioned media it is not necessary to wash the cells prior to commencing the R N A extraction procedure. For analysis of STC m R N A levels by Northern blotting, a sufficient amount of total R N A can be 5 obtained from 5 χ 10 cells to yield a good signal after a 1-2 day exposure to film. Acknowledgements. We would like to acknowledge the support of Dr. Henry G. Friesen, M D , Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada, in whose laboratory the primary culture system for trout CS cells was originally developed. We would also like to thank Ewa Jaworski for her invaluable technical assistance and the Natural Sciences and Engineering Council of Canada for financial support (GFW).
TV. References 1. Chomczynski, P. and N. Sacchi. Single-step method of R N A isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analyt. Biochem. 162: 156-159, 1987. 2. Gellersen, Β., G.F. Wagner, D.H. Copp, and H.G. Friesen. Development of a primary culture system for rainbow trout corpuscles of Stannius and characterization of secreted teleocalcin. Endocrinology 123: 9 1 3 - 9 2 1 , 1988. 3. Leibovitz, A. The growth and maintenance of tissue-cell culture in free gas exchange with the atmosphere. Am. J. Hygrol. 78: 173-180, 1963. 4. Wagner, G.F., B. Gellersen, and H.G. Friesen. Primary culture of teleocalcin cells from rainbow trout corpuscles of Stannius: regulation of teleocalcin secretion by calcium. Mol. Cell. Endocrinol. 62: 3 1 - 3 9 , 1989. 5. Weil, C , P., Hansen, D . Hyam, F. Le Gac, B. Breton, and L.W. Crim. U s e of pituitary cells in primary culture to study the regulation of gonadotropin hormone (GtH) secretion in rainbow trout: Setting up and validating the system as assessed by its responsiveness to mammalian and salmon gonadotropin releasing hormone. Gen. Comp. Endocrinol. 62: 202-209, 1986. 6. Wolf, K. and M.C. Quimby. Amphibian cell culture: Permanent cell line from the bullfrog (Rana catesbeiana). Science 144: 1578-1580, 1964.
and Mommsen
(eds.), Biochemistry
and molecular
biology of fishes, vol. 3
© 1994 Elsevier Science B.V. All rights reserved. C H A P T E R 23
Primary culture of rainbow trout corpuscles of Stannius BIRGIT GELLERSEN AND GRAHAM F. W A G N E R * Institute for Hormone and Fertility Research, Grandweg 64, 2000 Hamburg 54, Germany, and * Department of Physiology, Faculty of Medicine, University of Western Ontario, London, Ontario, Canada N6A 5C1
I. II.
Introduction Preparation of cell cultures 1. Harvesting of corpuscles of Stannius 2. Cell preparation III. Comments 1. Cell preparation 2. Choice of media 3. Culture conditions 3.1. Temperature 3.2. Osmotic pressure 3.3. Calcium content 4. Handling and storage of conditioned media IV. R N A extraction from plated CS cells Acknowledgements V. References
/.
Introduction
The corpuscles of Stannius (CS) are the source of stanniocalcin (STC), a hormone that is secreted in response to hypercalcemia and acts to inhibit calcium transport across the fish gill. These glands are ideally suited for primary culture, as they are readily accessible in most species and can be obtained in quantity to yield large numbers of cells. In this chapter, we have provided a detailed procedure for preparing and maintaining primary cultured rainbow trout (Oncorhynchus mykiss) CS cells. For those who are so inclined, we offer encouragement by stressing the ease with which these cells can be maintained in culture, with a minimum amount of expense, equipment and practice. We have also included media recipes for two different styles of culture, one requiring gas ( C O 2 ) and another that does not. Both techniques work well as we have used them both. The first technique employs commercially available D M E M or amphibian media, and requires a minimal amount of gassing and a cold cabinet kept at 10-15°C. This
274
Β. Gellersen and G.E
Wagner 2
is the method we used to set up our first primary cultures of trout CS cells . The advantage of this method is that it doesn't require an incubator and is therefore a low-cost approach. The main disadvantage of the method is that the cultures have to be monitored daily to ensure pH stability. It also requires a cold cabinet or similar environment maintained at a constant, low temperature (10-15°C). Therefore, either a refrigerator or a cold room would have to be dedicated exclusively to culturing CS cells. Alternatively, this method can be adapted directly for use with a C O 2 incubator. The second technique employs Leibovitz media (L-15), makes use of an incuba tor maintained at 10-15°C in a 4°C cold room and does not require gas. Hence, the incubator is used only for maintaining sterility and a stable, low temperature. The disadvantage of this method is that it requires an incubator and therefore entails extra costs. However, if an incubator can be obtained at little or no expense (an old C O 2 incubator will suffice), then this is the method of choice. The cultures require little attendance except for occasional feeding, pH stability is not an issue and most if not all institutions have a 4°C cold room. This is the method we now employ with excellent results.
77. Preparation of cell cultures 1. Harvesting of corpuscles of Stannius (day 1) Have the following materials at hand: (a) Sterile dissecting instruments (fine forceps, scissors, scalpels, alcohol lamp, 70% ethanol) (b) 50 ml tissue culture tubes with ca. 20 ml transport media on ice Kill fish with a blow to the head, slit the gills and drain the blood from the carcass by holding the animal by the tail in a vertical position. Then cut open the abdomen in a forward direction from the vent and remove the viscera and swim bladder. Usually there are three or four CS (small cream-coloured glands) in trout, midway along the kidney attached to the ventral margins or buried in and protruding from the dorsal surface of the kidney. Pierce the thin sheath of connective tissue covering the kidneys with fine forceps, remove the CS and place them in ice-cold transport media. Rinse forceps in 70% ethanol and pass them over a flame after each dissection. If glands are required from a large number of fish, an alternative procedure used at some Canadian fish hatcheries and modified for our own use is more expedient. A 1 χ 0.4 meter sheet of wood or plexiglass with 6-10 nails protruding from the front surface (along the top) is secured in a near-verticle position, preferably over a sink basin. After stunning each fish, impail them by the tail on the nails such that they hang vertically, head downward. With the fish secured in this manner, the bleeding, evisceration and dissection procedures can be done by one person. We routinely use this procedure to obtain glands from 40-50 fish in 2 - 3 hours. Keep the glands in transport media on ice until returning to the laboratory.
Primary culture of rainbow trout corpuscles of Stannius
275
2. Cell preparation Cell (a) (b) (c) (d) (e) (f) (g) (h) (i) (j)
preparation (day 1). Have the following materials at hand in the laboratory: sterile forceps, scalpels and 21 gauge needles, sterile glass petri dishes on ice, sterile Pasteur pipets (e.g. plastic liquipettes), 50 ml conical tissue culture centrifuge tubes, bench top centrifuge, sterile pipets and pipet tips, verticle turntable or shaker in a cold room, hemocytometer, 2 tissue culture vessels such as 24-well plates, 25-cm flasks, etc., a dissecting microscope.
All procedures involved in preparing the cell suspension are done in a sterile, laminar flow hood, keeping the tissue and solutions at 4°C whenever possible: Step 1. Decant glands from transport tube into chilled petri dish, rinse twice with transport media by aspirating wasted media with a pasteur pipet. Step 2. To drained CS, add a few ml of dissecting media and remove residual renal and fat tissue with fine forceps. Step 3. Transfer clean glands to a new petri dish containing a few ml of dissecting media. Step 4. Tear glands into small pieces with fine forceps. Step 5. Transfer suspension to 50-ml tube, spin 8 min at 1000 rpm (250 g). Step 6. Save pellet, transfer supernatant to new 50-ml tubes, sediment as above, discard supernatant, save second pellet. Step 7. Add 10 ml of trypsin solution to each of the two tubes. Step 8. Close tubes tightly and attach to a slow moving turntable in the cold room for overnight (16 h) digestion at 4-8°C (alternatively, place tubes in a horizontal position in a styrofoam box filled with ice and agitate on a shaker). Cell preparation (day 2). Step 1. Add 50 μΐ DNase (5 /xg/ml final cone.) per 10 ml of trypsinized cells. Step 2. Agitate 15 min at room temperature in a horizontal position. Step 3. Decant suspension into petri dish, tease apart undigested tissue with nee dles or fine forceps to aid dissociation of cells, remove undigested tissue fragments. Step 4. To break up cell clumps draw the cell suspension repeatedly through a 10 ml pipette, all the time pressing the pipette tip against the bottom of the petri dish. Step 5. Filter cell suspension with a Falcon cell strainer into a 50 ml centrifuge tube, centrifuge cells for 8 min at 1000 rpm. Step 6. Save pellet, transfer supernatant to new tube, sediment again as above, discard supernatant. Step 7. Wash both pellets with 10 ml of dissection media, centrifuge 8 min at 1000 g, discard supernatant. Step 8. Pool pellets after resuspension of cells in culture media (use 10 ml media for CS obtained from 10 fish).
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TABLE 1 Stock media formulations for gassed cultures (to make 1000 ml each) RPMI-1640 Stock with reduced NaHC03 - dissolve powder (without N a H C 0 3 , with L-Glutamine-Gibco) in 900 ml H 2 O - add 0.76 g N a H C 0 3 (to give final cone, of 9 mM) - adjust pH to 7.2 with 1 Ν HCl - make up to 1 liter, filter sterilize, keep at 4°C DM EM -
Stock (Dulbecco's Modified Eagle Medium with reduced NaHCOj dissolve powder (without N a H C 0 3 , with L-Glutamine-Gibco) in 900 ml H 2 O add 2.45 g N a H C 0 3 (to give final cone, of 30 mM) adjust pH to 7.2 with 1 Ν HCl make up to 1 liter, filter sterilize, keep at 4°C
MEM Stock (Minimal Essential Medium with Earle's Salts, with L-Glutamine-Gibco) Ready-to-use, keep at 4°C (contains 2.20 g N a H C 0 3 / L , 6.80 g NaCl/L, 0.20 g CaCl 2/L) EBSS Stock (Earle's Buffered Salt Solution) without NaCl 0.20 g C a C l 2 0.40 g KCl 0.20 g M g S 0 4 7 H 2 0 2.20 g N a H C 0 3 0 . 1 4 g N a H 2P O 4H 2O dissolve in 900 ml H 2 0 , adjust pH to 7.2, make up to 1 liter, filter sterilize, keep at 4°C HBSS Stock (Hanks' Balanced Salt Solution, with increased NaHCOj) - dissolve powder (without N a H C 0 3- G i b c o ) , in 900 ml H 2 0 - add 0.76 g N a H C 0 3 (to give 9 mM final cone.) - adjust pH to 7.2 with 1 Ν HCl - make up to 1 liter, filter sterilize, keep at 4°C
Step 9. To determine cell yield and viability, mix 20 μΐ cell suspension and 20 μΐ Trypan blue, wait 10 minutes, count viable cells (unstained) and dead cells (blue), do not count the RBC. 5 Step 10. Dilute cell suspension to 5 χ 10 cells/ml in media of choice and plate 1 2 ml/well in 24-well plates or 8 ml in 25-cm flasks. Gassed cultures. If a C O 2 incubator is unavailable, place cultures in tightly sealed containers. Purge the containers with 5% COil9S% air and maintain them in a cold cabinet (10-15°C). Domestic, plastic containers with water-tight lids are perfectly adequate for maintaining the cultures in a sealed environment. Inlet and outlet ports for purging containers with the CC^/air mixture can be improvised; stopcocked syringe needles can be inserted through the lids and secured in place with adhesive. Supplement gas to the sealed cultures daily, or when the phenol red in the media indicates a pH above 7.5. Chambers which are specifically designed for sealed cultures are commercially available (Caltech Scientific). Alternatively, if a C O 2 incubator is available, place it in a cold room and maintain the cells at 10-15°C, supplemented with a 5% C 0 2 / 9 5 % air mixture. Non-gassed cultures. In the case of cells plated in L-15 media, maintain cultures in a 10-15°C cold cabinet (sealed containers are not required) or an incubator in
Primary culture of rainbow trout corpuscles of Stannius
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a 4°C cold room, maintained at 10-15°C. Be aware of the fact that cold rooms are notoriously unclean environments. Therefore, clean and rinse all inner surfaces of the incubator with 70% ethanol prior to preparing each new lot of cells. Cell preparation (day 4). Check cultures under an inverted microscope. The cells should have flattened out and attached to the plates, but secretory granules will not be evident at this point. There will also be a lot of cellular debris in the cultures. Aspirate off media with gentle suction, wash cells with HBSS or culture media minus serum and antibiotics, aspirate and replace with fresh media. Cell preparation (day 7). Secretory granules should now be clearly visible in the cells, as illustrated in Panels 3b-6b, and the cultures are ready for experimental manipulation.
III.
Comments
1. Cell preparation The removal of kidney and fat tissue from the glands is best done under a dissecting microscope. The proportion of viable cells after the cold trypsinization should be 6 2 7 5 - 9 5 % with a yield of 1.4-2.4 χ 1 0 viable cells/gland in 2.5 kg trout and 1 6 2 χ 10 cells/fish in 0.5 kg trout (the number and size of individual glands are highly variable between fishes of the same weight). In our experience, it is most convenient to collect the glands on a Tuesday morning (day 1), such that the cells have formed monolayers by Friday (day 4). After washing the cells on day 4, they can be left over the weekend and are ready for experimentation on the following Monday (day 7). 2. Choice of media For gassed cultures, both D M E M and amphibian media work equally well, so choose your media on the basis of cost and availability. RPMI is only required for experiments involving manipulations of media calcium content. D o not prepare or plate cells in RPMI as the calcium content is too low. This may affect cell viability and the cells will take too long to attach to the plates. HEPES buffer is added to stabilize the pH at 7.4-7.5 in the sealed, gassed cultures when a C O 2 incubator is unavailable for use. Nonetheless, occasional gassing will be required to lower the p H and therefore it is best to check the cultures daily. For nongassed cultures, Leibovitz media can be prepared in the laboratory with considerable savings over commercially available media. Feed cells with fresh media at least twice weekly. 3. Culture
conditions
Three variables deserve special interest regarding culture conditions for rainbow trout CS cells: temperature, osmotic pressure and calcium concentration.
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TABLE 2 Media preparation for gassed cultures Prepare fresh from the stock solutions listed in Table 1 before harvesting glands Transport medium 96 ml HBSS stock 2 ml H E P E S (20 mM final cone.) 1 ml Penicillin-Streptomycin (100 U/ml-100 /zg/ml final cone.) 1 ml Fungizone (2.5 μg/ml final cone.) Dissection medium 98 ml transport medium 2 ml B S A (0.2% final cone.) Trypsin solution 18 ml transport medium 0.4 ml B S A (0.2% final cone.) 2 ml Trypsin (0.25% final cone.) Amphibian culture medium 50 ml whole egg ultrafiltrate (10% final cone.) 50 ml FCS (10% final cone.) 125 ml EBSS/NaCl-free stock (25% final cone.) 275 ml MEM stock (55% final cone.) 10 ml H E P E S (20 mM final cone.) 2.5 ml Penicillin-Streptomycin DMEM culture medium 440 ml D M E M stock 50 ml FCS (10% final cone.) 10 ml H E P E S (20 mM final cone.) 2.5 ml Penicillin-Streptomycin RPMI serum-free culture medium (RPMISF) 97 ml RPMI-1640 stock 2 ml H E P E S 0.5 ml Penicillin-Streptomycin 1 ml B S A ( 0 . 1 % final cone.)
*
* Only for experiments requiring low calcium levels.
3.1. Temperature In our view, it is best to culture trout CS cells at 10-15°C to mimic the natural environment. It may also be possible to maintain the cells at room temperature, however we have not tried this. Furthermore, what effects this might have on CS cell metabolism is unknown. Cultures can be maintained "stored" in the fridge without attendance for several weeks. 3.2. Osmotic pressure Rainbow trout inhabit both seawater and fresh water and can therefore adapt to a wide range of salinities. Trout plasma is approximately 300 mOsm, whereas mammalian culture media such as D M E M and RPMI-1640 have values of 2 7 0 275 mOsm, corresponding to mammalian plasma. Nevertheless, trout CS cells do well in these media. The preparation of HBSS buffer with increased NaHCOß, as 5 described in the media formulations, gives a value of 300 mOsm . Interestingly,
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TABLE 3 Stock media and media formulations for nongassed cultures Leibovitz Stock (L-15) with L-glutamine and without NaHCOj (Gibco), - ready to use, keep at 4°C 3 - or prepare according to original recipe (Leibovitz 1963) Transport medium 98 ml L-15 stock 1 ml Penicillin-Streptomycin (100 U/ml-100 ^g/ml final cone.) 1 ml Fungizone (2.5 ^g/ml final cone.) Dissection medium 98 ml transport medium 2 ml B S A (0.2% final cone.) Trypsin solution 18 ml transport medium 0.4 ml B S A (0.2% final cone.) 2 ml Trypsin (0.25% final cone.) Leibovitz culture medium (L-15) 90 ml L-15 stock 10 ml Fetal Bovine Serum 0.5 ml Penicillin-Streptomycin Leibovitz serum-free culture medium (L-15-SF) 99 ml L-15 stock 1 ml B S A 0.5 ml Penicillin-Streptomycin
TABLE 4 Common stock solutions FCS (Fetal Calf Serum): store 50 ml aliquots at - 2 0 ° C * Whole Egg Ultrafiltrate
(Gibco)
BSA (Bovine Serum Albumin Fraction V, cell culture tested) (Sigma). Prepare 10% stock solution, pH 7.4, in HBSS or L-15 medium. Filter sterilize, freeze aliquots Trypsin (Gibco): Ready-to-use 10X stock solution (2.5% in 0.9% NaCl), keep aliquots at - 2 0 ° C Penicillin-Streptomycin (Gibco): Ready-to-use stock solution with 10,000 U Penicillin/ml and 10 mg Streptomycin/ml; keep aliquots at —20°C Fungizone (Gibco): Freeze aliquots of a stock solution containing 250 ^ g Amphotericin B/ml * HEPES (Gibco): Ready-to-use 1 Μ stock solution, keep at 4°C DNase I (Boehringer): Prepare a 1 mg/ml stock solution in phosphate buffered saline, filter sterilize, freeze aliquots Trypan Blue (Boehringer): ready-to-use 0.5% solution in 0.9% NaCl, keep at room temperature * Not required for nongassed cultures
6
Amphibian media which was originally developed for bullfrog cultures is 242 mOsm, but also supports long term maintenance of CS cells. Due to its low osmotic pressure, amphibian media is applicable to experiments designed to test the effects of increasing osmotic pressure.
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Fig. 1. Primary cultured rainbow trout CS cells viewed under brightfield (a) and phase contrast microscopy (b) six days after initial plating in L-15-SF medium (1.2 mM calcium). Panels 1-6 are of CS cells following a one day exposure to L-15 medium containing different concentrations of ionic calcium. Panel la,b. CS cells in 0.3 mM calcium. N o t e the rounded appearance of the cells (arrows in a,b). Panel 2a,b. CS cells in 0.7 mM calcium. N o t e that cells are less rounded (arrows in b). Panel 3a,b. CS cells in 1.1 mM calcium. The cells have flattened and are filled with secretory granules (arrows in b).
3.3. Calcium content For efficient attachment of CS cells after plating, culture media with a "regular" calcium content are recommended. Amphibian and D M E M media when formulated as described, contain 1.67-1.8 mM total C a 2 +, the ionic (free) C a 2 + concentration being somewhat lower due to binding of calcium to serum proteins (Amphibian medium has 1.34 mM ionic C a 2 +) . For experiments designed to test the effects of calcium on STC secretion from CS cells, a media with a low calcium content such as RPMI is desirable. For a limited period of time, CS cultures can be
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Fig. 1 (continued). Panel 4a,b. CS cells in 1.5 mM calcium. The density of cells containing secretory granules (arrows in b) is not noticeably different than cells in 1.1 mM calcium. Panel 5a,b. CS cells in 1.9 mM calcium. Fewer cells contain secretory granules (arrows in b) in comparison to cells in Panels 3 and 4. Panel 6a,b. CS cells in 2.3 mM calcium. Very few cells now contain secretory granules (arrows in b).
maintained in RPMI-SF which has 0.32 mM free C a 2 + (ref. 4). For experiments on the effects of calcium employing L-15 media, we prepare our own stock media using the original formulation, except that calcium content is adjusted to 0.3 mM. In this way, calcium can then be added back to the plated cells to obtain any desired concentration. After initial plating in L-15 media containing normal calcium concentrations (1.2 mM), trout CS cells can then be maintained for at least a week in L-15 containing only 0.3 mM calcium. However, the morphology of the cells changes under these conditions (they round up) and only return to normal (a flat monolayer) when calcium levels are restored (Fig. 1, panels la,b and 3a,b).
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4. Handling and storage of conditioned
Wagner
media
Store conditioned media at - 7 0 ° C and thaw on ice. After thawing, the media should be vortexed and then centrifuged at 12,000 g to pellet cellular debris prior to analysis.
IV RNA extraction from plated CS cells 1
To isolate total R N A from plated cells, use the method of Chomczynski and Sacchi as described. However, after aspiration of conditioned media it is not necessary to wash the cells prior to commencing the R N A extraction procedure. For analysis of STC m R N A levels by Northern blotting, a sufficient amount of total R N A can be 5 obtained from 5 χ 10 cells to yield a good signal after a 1-2 day exposure to film. Acknowledgements. We would like to acknowledge the support of Dr. Henry G. Friesen, M D , Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada, in whose laboratory the primary culture system for trout CS cells was originally developed. We would also like to thank Ewa Jaworski for her invaluable technical assistance and the Natural Sciences and Engineering Council of Canada for financial support (GFW).
TV. References 1. Chomczynski, P. and N. Sacchi. Single-step method of R N A isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analyt. Biochem. 162: 156-159, 1987. 2. Gellersen, Β., G.F. Wagner, D.H. Copp, and H.G. Friesen. Development of a primary culture system for rainbow trout corpuscles of Stannius and characterization of secreted teleocalcin. Endocrinology 123: 9 1 3 - 9 2 1 , 1988. 3. Leibovitz, A. The growth and maintenance of tissue-cell culture in free gas exchange with the atmosphere. Am. J. Hygrol. 78: 173-180, 1963. 4. Wagner, G.F., B. Gellersen, and H.G. Friesen. Primary culture of teleocalcin cells from rainbow trout corpuscles of Stannius: regulation of teleocalcin secretion by calcium. Mol. Cell. Endocrinol. 62: 3 1 - 3 9 , 1989. 5. Weil, C , P., Hansen, D . Hyam, F. Le Gac, B. Breton, and L.W. Crim. U s e of pituitary cells in primary culture to study the regulation of gonadotropin hormone (GtH) secretion in rainbow trout: Setting up and validating the system as assessed by its responsiveness to mammalian and salmon gonadotropin releasing hormone. Gen. Comp. Endocrinol. 62: 202-209, 1986. 6. Wolf, K. and M.C. Quimby. Amphibian cell culture: Permanent cell line from the bullfrog (Rana catesbeiana). Science 144: 1578-1580, 1964.