This project was funded by the Norwegian University of Life Sciences and the Research Council of Norway, grant number 243811 and 244461 (Aquaculture program). We are grateful to Lourdes Carreon Tan at the Norwegian University of Life Sciences for maintaining the fish facility.

The animal experiments performed in this study were approved by the Norwegian University of Life Sciences, following guidelines for the care and welfare of research animals.
1. Preparation of Solutions
<ol>
	<li>Calibrate the osmometer and pH meter instruments according to the manufacturer&#8217;s instructions to ensure correct measurements.</li>
	<li>Prepare 500 mL of Ca2+- and Mg2+-free Dulbecco&#8217;s phosphate-buffered saline (dPBS), adjusting the pH to 7.75 (step 1.2.1) and the osmolality to 290 mOsm/kg (step 1.2.2) before a sterile filtration (step 1.2.3).
	<ol>
		<li>Adjust the pH to 7.75 with a calibrated pH meter by carefully adding drops of a 1 M NaOH solution while carefully stirring the solution.</li>
		<li>Measure the osmolality of the solution using a calibrated osmometer and calculate the amount of mannitol needed to increase the osmolality to 290 mOsm/kg. Add the required amount of mannitol and then measure the osmolality of the solution again to ensure the correct adjustment. 
		NOTE: The amount of mannitol needed depends on the osmolality measured, which usually varies between different batches. One molecule of mannitol equals 1 osmotic particle.</li>
		<li>Sterile-filter the dPBS solution using a 0.2 &#181;m filter. 
		NOTE: The solution may be stored at 4 &#176;C for at least 3 months.</li>
	</ol>
	</li>
	<li>Prepare 500 mL of Leibovitz (L-15) culture medium without L-glutamine and bicarbonate, and supplement it with 10 mM NaHCO3 (step 1.3.1), 4.5 mM glucose (step 1.3.2), and 2 mM commercially available glutamine (step 1.3.3). Adjust the solution to 290 mOsm/kg (step 1.3.4), sterile-filter it (step 1.3.5), and add 2.5 mL of a penicillin-streptomycin solution (step 1.3.6). 
	NOTE: The solution may be stored at 4 &#176;C for at least 4 weeks.
	<ol>
		<li>Add 420 mg of NaHCO3 per 500 mL of culture medium to achieve a 10 mM solution.</li>
		<li>Add 405 mg of glucose per 500 mL of culture medium to achieve a 4.5 mM solution.</li>
		<li>Add 5 mL of 100x stock of a glutamine solution in 500 mL of culture medium to achieve a 2 mM solution. Stir until all solutes are dissolved.</li>
		<li>Adjust the solution to 290 mOsm/kg with mannitol using an osmometer (as performed in step 1.2.2).</li>
		<li>Perform a sterile filtration of the L-15 medium using a 0.2 &#181;m filter.</li>
		<li>After the sterile filtration, add 2.5 mL of a penicillin-streptomycin solution, corresponding to 50 U/mL of penicillin and 50 &#181;g/mL of streptomycin.</li>
	</ol>
	</li>
	<li>Prepare 50 mL of a trypsin solution using 1 mg/mL of (0.1%) trypsin type II-S dissolved in the modified dPBS (prepared in step 1.2). Make aliquots of 2 mL in sterile plastic tubes and store them at -20 &#176;C until use. 
	NOTE: Aliquots of 2 mL can be stored at -20 &#176;C for at least 3 months.</li>
	<li>Prepare 50 mL of a trypsin inhibitor solution using 1 mg/mL of (0.1%) trypsin inhibitor type I-S, and supplement it with 2 &#181;g/mL of DNase I dissolved in the modified dPBS (prepared in step 1.2). Make aliquots of 2 mL in sterile plastic tubes and store them at -20 &#176;C until use. 
	Note: Aliquots of 2 mL can be stored at -20 &#176;C for at least 3 months.</li>
</ol>
2. Preparation of Equipment
<ol>
	<li>Prepare glass pipettes by fire&#160;polishing the tip for the cell dissociation. Make tips with 2 different size categories (a medium opening of 0.6&#8211;0.8 &#181;m and a small opening of 0.4&#8211;0.6 &#181;m) by playing with the time and the distance from the flame while simultaneously turning the pipette. Place the glass pipettes in a clean glass container and autoclave them (using the solid mode at 105 &#176;C for 45 min).</li>
	<li>Prepare a polystyrene box with ice.</li>
	<li>Prepare a plastic tube with 1.5 mL of modified dPBS (prepared in step 1.2) to transfer the pituitaries to during the dissection and keep it on ice.</li>
	<li>Prepare a 15 mL tube with 10 mL of modified dPBS (prepared in step 1.2) and keep it on ice until the cell dissociation.</li>
	<li>Prepare fresh or thaw aliquots of the trypsin and trypsin inhibitor solutions (prepared in steps 1.4 and 1.5) and keep them on ice until use.</li>
	<li>Set the water bath to 26 &#176;C before starting the dissection to allow the water to reach the desired temperature before the chemical dissociation of the pituitaries.</li>
	<li>Clean all dissection tools with 70% ethanol before, during, and after use, including fine forceps to use for the dissection and fine needles and a wax plate to pin the fish. Use clean gloves (change and clean frequently) during the entire procedure to avoid a potential contamination of the cells.</li>
</ol>
3. Pituitary Dissection and Cell Dissociation
<ol>
	<li>Euthanize the fish by immersing it in ice slush (0 &#176;C). Leave the fish in the ice slush for at least 1 min to ensure an irreversible hypothermic shock. 
	NOTE: Immobilization and equilibrium loss will occur after a few seconds. Make sure the fish is completely submerged in the ice water and does not lie on top of the ice, as the latter will lead to suffocation and potential skin burns instead of a rapid and humane hypothermic shock.</li>
	<li>Quickly transfer the fish in a net to a wax plate under the dissection microscope and destroy the spine by a needle punch through the neck.</li>
	<li>Pin the head of the fish to the wax plate using fine needles, by inserting one needle in the front (over the mouth) and one on each side of the head (behind the brain) to stabilize the head before the dissection.</li>
	<li>View the fish under the dissection microscope and gently scrape off the scales at the top of the head using fine forceps with an angled tip.</li>
	<li>Carefully insert the angled tip of the fine forceps under the skin from the side of the head and remove the skull roof by slowly moving the forceps in the direction of the mouth, while holding a firm grip to the forceps enclosing the skull roof.</li>
	<li>Cut the spinal cord completely off using forceps with an angled tip. 
	NOTE: Time is an important factor, so work efficiently and accurately to limit the time spent from the dissection of the pituitaries until the cells are plated in the dish. After some practice, pituitary dissection should take around 2&#8211;3 min per fish, of which only around 10 s are needed to pick the pituitary when the brain is already exposed.</li>
	<li>Carefully flip the brain over towards the mouth to expose the pituitary and dissect it with clean forceps with a straight tip. Transfer the pituitary to a plastic tube containing 1.5 mL modified dPBS (for details, see step 2.3) placed on ice.&#160;Check the tip of the forceps under the microscope to ensure that the pituitary is successfully added to the tube and nothing is left on the forceps. 
	NOTE: Dissect as many pituitaries as needed, depending on the number of culture dishes that are going to be prepared simultaneously. As a rule of thumb, calculate at least 10 pituitaries from adult fish per dish to have an appropriate density of cells (which depends on the downstream application).</li>
	<li>Spin down the pituitaries in a tabletop centrifuge for 1&#8211;2 s at room temperature. Carefully remove the dPBS using a glass pipette and take care to avoid removing any pituitaries.</li>
	<li>Add 1 mL of the trypsin solution (prepared in step 1.4) to wash the pituitaries, spin them down in a tabletop centrifuge for 1&#8211;2 s, and remove most of the liquid using a glass pipette before adding an additional 1 mL of trypsin solution.</li>
	<li>Incubate the tube in a water bath at 26 &#176;C for 30 min, preferably with gentle shaking, or alternatively, flick the tube a couple of times during the incubation period.</li>
	<li>Spin down the pituitaries in a tabletop centrifuge for 1&#8211;2 s at room temperature and make sure all pituitaries are located at the bottom of the tube. Remove most of the trypsin solution with a glass pipette and add 1 mL of the trypsin inhibitor solution (prepared in step 1.5) to inactivate the trypsin and wash the pituitaries.</li>
	<li>Collect the pituitaries at the bottom of the tube by spinning them down in a tabletop centrifuge for 1&#8211;2 s at room temperature. Remove most of the liquid with a glass pipette and add 1 mL of the trypsin inhibitor solution (prepared in step 1.5). Place the tube on a water bath at 26 &#176;C for 20 min, preferably with gentle shaking, or alternatively, flick the tube a couple of times during the incubation period to inactivate the trypsin.</li>
	<li>Prepare a 35 mm poly-d-lysine-coated cell culture dish with a central glass bottom by adding 2 mL of L-15 medium and letting that incubate for at least 10 min at 26 &#176;C and with 1% CO2 so that it can reach the right temperature and pH before plating the cells. 
	NOTE: A plastic cell culture dish can also be used. The main advantage of using a dish with a centralized glass bottom is that the area where the cells are plated is smaller, and therefore, fewer pituitary cells are required per dish. Some downstream applications might also require a glass bottom (i.e., some imaging techniques).</li>
	<li>Set the cooling centrifuge for the 15 mL tubes to 4 &#176;C to allow it to reach the desired temperature before starting the mechanical dissociation.</li>
	<li>Collect the pituitaries at the bottom of the tube by spinning down the tube with the trypsin inhibitor solution (from step 3.11) in a tabletop centrifuge for 1&#8211;2 s at room temperature and make sure all pituitaries are located at the bottom of the tube before carefully removing most of the trypsin inhibitor solution using a glass pipette. 
	Note: Perform all the following steps of the protocol in a laminar flow bench (LAF) certified for cell work to avoid a contamination of the cells.</li>
	<li>Add 1 mL of ice-cold modified dPBS (prepared in step 2.4) to the pituitary tissue pieces and gently suck the tissue pieces up and down (6&#8211;7x) in a fire-polished glass pipette (prepared in step 2.1). 
	NOTE: Temperature is an essential aspect, so keep all solutions at 4 &#176;C. From this point onward, perform all steps on ice when possible.</li>
	<li>Spin down the tissue pieces in a tabletop centrifuge for 1&#8211;2 s at room temperature and carefully transfer the upper part of the dPBS containing the dissociated cells (almost 1 mL) to a new 15 mL tube placed on ice. Avoid moving the pipette towards the bottom of the tube where the tissue pieces are left.</li>
	<li>Repeat step 3.16&#8211;3.17, dissociating the cells in 1 mL of ice-cold dPBS at a time, until all 10 mL of dPBS is used. Start using a glass pipette with a medium-sized opening and switch to a glass pipette with a smaller opening towards the end (for the last 3&#8211;4 mL of dPBS added) of the dissociation. If there are still visible tissue pieces towards the end of the dissociation procedure, increase the pipetting to approximately 10x towards the 2 last steps of dPBS for dissociation, and finally leave the residual tissue pieces behind. Resuspend the cells gently and avoid creating bubbles as it may cause cell damage when the cells attach to the surface of the bubbles. 
	NOTE: This is a critical step of this protocol and requires some training to achieve a good result.</li>
	<li>Balance the tube in the centrifuge (pre-cooled to 4 &#176;C) and spin down the cells at 200 x g for 10 min at 4 &#176;C. Make sure to mark the side of the tube where the cell pellet will be.</li>
	<li>Carefully remove the tube from the centrifuge and gently transfer the supernatant with a glass pipette to an empty 15 mL plastic tube directly after the centrifugation. Be sure to remove most of the supernatant but take care to avoid losing the small (often invisible) cell pellet.</li>
	<li>Carefully resuspend the cell pellet in 50&#8211;100 &#181;L of L-15 culture medium. 
	NOTE: At this step, it is possible to count the cells and/or perform a viability test (such as a cell counting in the presence of trypan blue using a hemocytometer), but beware that using part of the cell suspension for this purpose will lower the yield. Avoid using a large suspension volume as it will increase the risk of cells diffusing outside of the center of the dish.</li>
	<li>Carefully drip the dissociated cells in the middle of the cell culture dish containing 2 mL of the L-15 medium (prepared in step 3.13). Allow the cells to sink to the bottom of the dish for approximately 5 min before moving them to the incubator, to avoid having the cells spread outside of the sunken part of the glass bottom.</li>
	<li>Incubate the dish at 26 &#176;C and 1% CO2 to allow all the cells to sink thoroughly and attach to the bottom of the dish.</li>
	<li>After 30 min, look at the cells in the microscope to make sure they have attached to the culture dish.</li>
	<li>Continue to incubate the cells at 26 &#176;C and 1% CO2.</li>
	<li>Carefully change most of (but not all) the medium every 3&#8211;4 days. 
	Note: Avoid changing the medium more frequently, as it may disturb the cells and make them detach from the glass bottom. Use the cells for experiments within a week after seeding them.</li>
</ol>

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The importance of osmolality, pCO2, and pH.</a> General and Comparative Endocrinology. 178 (2), 206-215 (2012).</li><li>Wittbrodt, J., Shima, A., Schartl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medaka+-+a+model+organism+from+the+far+East.">Medaka - a model organism from the far East.</a> Nature Reviews Genetics. 3, 53-64 (2002).</li><li>Matsuda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMY+is+a+Y-specific+DM-domain+gene+required+for+male+development+in+the+medaka+fish.">DMY is a Y-specific DM-domain gene required for male development in the medaka fish.</a> Nature. 417 (6888), 559-563 (2002).</li><li>Kasahara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+medaka+draft+genome+and+insights+into+vertebrate+genome+evolution.">The medaka draft genome and insights into vertebrate genome evolution.</a> Nature. 447 (7145), 714-719 (2007).</li><li>Miyanishi, H., Inokuchi, M., Nobata, S., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Past+seawater+experience+enhances+seawater+adaptability+in+medaka,+Oryzias+latipes.">Past seawater experience enhances seawater adaptability in medaka, Oryzias latipes.</a> Zoological Letters. 2 (12), (2016).</li><li>Waymouth, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmolality+of+mammalian+blood+and+of+media+for+culture+of+mammalian+cells.">Osmolality of mammalian blood and of media for culture of mammalian cells.</a> In Vitro. 6 (2), 109-127 (1970).</li><li>Cameron, J. 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The authors have nothing to declare.

In vitro cell culture systems provide powerful tools for researchers to answer a plethora of different biological questions if used in the right way1. It is important to remember that dissociated cells that have lost their connections to the neighboring cells may have obtained different functional properties than they originally had in vivo. To avoid being at the risk of misinterpreting the results obtained from in vitro experiments, it is important to consider adjusting...

Cell culture is one of the main tools used in molecular biological research, providing an excellent model system for answering different biological questions ranging from normal cellular physiology to drug screening and carcinogenesis1. Primary cells, isolated directly from the animal tissue using enzymatic and/or mechanical methods,are often considered more biologically relevant than cell lines as the biological response may be closer to the in vivo situation. Protocols for preparing primary cell cultures should be optimized for each species and cell type of interest in order to mimic the characteristics to which a cell is adapted and obtain physiologically meaningful results.
Numerous protocols describe culture conditions for mammalian cell systems, while similar protocols describing primary culture conditions for fish cells are rather scarce in comparison. Cells are vulnerable to rapid changes in temperature, pH, and osmolality, and are particularly fragile during the dissociation procedure. Commercial salt solutions and culture media used for mammalian cell cultures are not optimal for teleost fish, especially in terms of pH buffer system(s) and osmolality. It is, therefore, important to measure and adjust the solutions to physiologically relevant levels of these parameters in the species of interest.
Primary pituitary cultures have been made from several teleost fish species, including common carp (Cyprinus carpio)2,3, grass carp (Ctenopharyngodon idella)4, goldfish (Carassius auratus)5, rainbow trout (Oncorhynchus mykiss)6, European eel (Anguilla anguilla)7, tilapia (Oreochromis mossambicus)8, zebrafish (Danio rerio)9, and Atlantic cod (Gadus morhua)10. Apart from adjusting the incubation temperature to the species of interest, several of these protocols have incubated the cells at mammalian-like conditions that may be suboptimal for the species of interest, with a pH from 7.2 to 7.5 in a humidified atmosphere containing 3 - 5% CO2. In addition, it is unclear if the osmolality of solutions used for preparing several of these primary cell cultures were adjusted and stable between different solutions.
The current protocol is based on previous work with primary cultures from Atlantic cod10 and comprises adjustments of incubation temperature, osmolality, pH, and pH buffer systems, including the partial pressure of carbon dioxide (pCO2), to the physiology of medaka (O. latipes). Medaka is a small (3&#8211;4 cm) freshwater fish, native to East Asia. These days, it is used as a model species in many research laboratories around the world, as it is relatively easy to breed and highly resistant to many common fish diseases11. There are several advantages of using medaka as a model, including a temperature tolerance from 4&#8211;40 &#176;C11, a short generation time, transparent embryos, a sex-determining gene12, and a sequenced genome13, as well as many other available genetic resources.
The primary culture conditions in this protocol are optimized to match the temperature of 26 &#176;C that medakas are kept at in the fish facility. Further, the osmolality is reduced from 320 mOsm/kg from Atlantic cod living in salt water to 290 mOsm/kg for medaka living in fresh water and is in accordance with the normal osmolality of medaka plasma14. In comparison, the typical osmolality of mammalian plasma is in the range of 275&#8211;295 mOsm15. Fish lives in a variety of temperatures and have gills that are in direct contact with water, making the pH and buffer capacity of the blood and extracellular fluid in fish different from those in mammals. Mammalian culture media usually include buffer systems that result in a pH of around 7.4 when the media are equilibrated to a standard atmosphere of 5% CO2 in humidified air at 37 &#176;C. The pH is temperature dependent and the value for neutral pH (in water) increases with a decreasing temperature16. Typical teleost fish plasma pH ranges from 7.7 to 7.917. The optimization of this protocol included a reduction from pH 7.85 for cod kept at 12 &#176;C to pH 7.75 for medaka kept at 26 &#176;C by increasing the CO2 from 0.5% to 1%.
In addition, the bicarbonate buffer capacity is quite different in fish and mammals. CO2 is easily exchanged over the gills in fish and the pCO2 in water is only a small fraction of the pCO2 in the lung18. Changing either the temperature or the pCO2 will change the pH and buffer of the medium. Consequently, neither the pH nor the pCO2 recommended for incubating mammalian cells is optimal for fish cells, and therefore, the culture media should be optimized with buffer systems containing physiologically relevant values for fish and the particular species of interest. This protocol describes how to prepare primary cell cultures from medaka pituitaries and include adjustments of the incubation temperature, osmolality, pH, and pH buffer system, in addition to other important parameters to consider when preparing primary cell cultures from non-mammalian species.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (dPBS), without calcium chloride and magnesium chloride</td><td>Sigma (Merck, Damstadt, Germany)</td><td>D8537</td><td>Adjust solution to pH 7.75 with 1M NaOH and 290 mOsm with mannitol.</td></tr><tr><td>L-15 medium (Leibovitz), witout L-glutamine</td><td>Sigma (Merck, Damstadt, Germany)</td><td>L5520</td><td>Supplement 500 ml culture medium with 10 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 4.5 mM glucose, 2 mM Glutamax. Adjust solution to 290 mOsm with mannitol and filter solution through a 0.2 &amp;micro;m PES sterile filter, before adding 2.5 mL Penicillin-Streptomycin solution (see below for details).</td></tr><tr><td>NaOH</td><td>Sigma (Merck, Damstadt, Germany)</td><td>S5881</td><td>Add drops of 1 M solution to increase pH of dPBS and culture medium to 7.75.</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma (Merck, Damstadt, Germany)</td><td>S5761</td><td>10 mM NaHCO&lt;sub&gt;3&lt;/sub&gt; equals 420 mg per 500 mL culture medium.</td></tr><tr><td>D-mannitol</td><td>Sigma (Merck, Damstadt, Germany)</td><td>63565</td><td>Use to increase osmolality of dPBS and culture medium. Calculate correct amount needed to reach an osmolality of 290 mOsm.</td></tr><tr><td>D-glucose</td><td>Sigma (Merck, Damstadt, Germany)</td><td>G5400</td><td>4.5 mM&amp;nbsp; D-glucose equals 405 mg per 500 mL culture medium.</td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco (Life Technologies, Paisley, UK)</td><td>35050-061</td><td>Alternative to L-glutamine, with increased stability. 2 mM Glutamax equals 5 ml of 100x stock in 500 mL culture medium.</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma (Merck, Damstadt, Germany)</td><td>P0781</td><td>Stock solution 10,000 units penicillin and 10 mg streptomycin per mL. Use 2.5 mL of stock solution in 500 mL L-15 medium (equivalent of 50 U/mL Penicillin and 50 &amp;micro;g/mL Streptomycin).</td></tr><tr><td>Trypsin type II-S</td><td>Sigma (Merck, Damstadt, Germany)</td><td>T7409</td><td>Prepare 1 mg/mL in dPBS solution.</td></tr><tr><td>Trypsin inhibitor type I-S</td><td>Sigma (Merck, Damstadt, Germany)</td><td>T6522</td><td>Prepare 1 mg/mL in dPBS solution, supplement with 2 &amp;micro;g/ml Dnase I (see details below).</td></tr><tr><td>Dnase I</td><td>Sigma (Merck, Damstadt, Germany)</td><td>D5025</td><td>Use in trypsin inhibitor solution (see above).</td></tr><tr><td>0.2 &amp;micro;m Polyethersulfone (PES) sterile filter system</td><td>Corning Inc. (Corning, NY)</td><td>431097</td><td>Use for sterile filtration of dPBS and L-15 medium after adjustments.</td></tr><tr><td>35 mm cell culture dish with glass bottom, poly d-lysine coated</td><td>MatTek Corporation (Ashland, MA)</td><td>P35GC-1.5-10-C</td><td>Can also be replaced by plastic dish, depending on downstream application.</td></tr><tr><td>Dumont #5 fine forceps</td><td>Fine Science Tools (CA)</td><td>11254-20</td><td>Straight tip</td></tr><tr><td>Dumont #5/45 fine forceps</td><td>Fine Science Tools (CA)</td><td>11253-25</td><td>Angled 45&amp;deg; tip</td></tr><tr><td>Stereo microscope SZ61</td><td>Olympus Corp. (Tokyo, Japan)</td><td></td><td>Use for dissection of pituitaries.</td></tr><tr><td>Alegra X-22R Centrufuge</td><td>Beckman Coulter Inc. (Brea, CA)</td><td></td><td>With cooling option (similar to current model XR-30).</td></tr><tr><td>Water bath</td><td>Techne (Staffordshire, UK)</td><td></td><td>Any water bath with the possibility of adjusting temperature will do.</td></tr><tr><td>Pasteur glass pipettes</td><td>VWR (NY)</td><td>612-1701</td><td>Outer diameter 1.6 mm, fire polish and autoclave before use.</td></tr><tr><td>Galaxy MiniStar table centrifuge</td><td>VWR (NY)</td><td>521-2844</td><td>Any small table centrigue will do.</td></tr><tr><td>Fine needles/insect pins</td><td>Fine Science Tools (CA)</td><td>26001-40</td><td>Diameter 0.03 mm. Other fine needles can be used instead.</td></tr><tr><td>Wax plate</td><td></td><td></td><td>Custom made by adding melted paraffin wax in large petri dish.</td></tr></tbody></table>

preparation of a high-quality primary cell culture from fish pituitaries

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment. Despite vast differences in the physiology between mammals and fish, primary cell culture protocols from fish are often based on mammalian culture conditions, often with only minor modifications. The environmental differences affect not only body temperature, but also blood serum parameters such as osmolality, pH, and pH buffer capacity. As cell culture media and similar working solutions are meant to mimic characteristics of the extracellular fluid and/or blood serum to which a cell is adapted, it is crucial that these parameters are adjusted specifically to the animal in question.
The current protocol describes optimized primary culture conditions for medaka (Oryzias latipes). The protocol provides detailed steps on how to isolate and maintain healthy dissociated pituitary cells for more than one week and includes the following steps: 1. the adjustment of the osmolality to the values found in medaka blood plasma, 2. the adjustment of the incubation temperature to normal medaka temperature (here in the aquarium facility), and 3. the adjustment of the pH and bicarbonate buffer to values comparable to other fish species living at similar temperatures. The results presented using the described protocol promote physiologically meaningful results for medaka and can be used as a reference guide by scientists making primary cell cultures from other non-mammalian species.

This protocol describes the preparation of a primary cell culture from medaka pituitaries and provides healthy cells that can be maintained in a culture for at least one week. The protocol is based on physiological relevant values for medaka14 and is additionally optimized to the pituitary tissue in adult fish, using a pH of 7.75 and an osmolality of 290 mOsm/kg during the entire procedure from the tissue harvesting to the plated cells in culture (<strong class="xf...

Watch this Scientific Journal Video about Preparation of a High-quality Primary Cell Culture from Fish Pituitaries at JoVE.com

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Here we describe a protocol to prepare and maintain primary pituitary cell cultures from medaka (Oryzias latipes). The optimized conditions in this protocol take important parameters such as temperature, osmolality, and pH into consideration by mimicking the physiological conditions of the fish, thereby enabling physiologically more meaningful results.

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled ...

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Research

JoVE Journal

Neuroscience

15.1K Views.  Norwegian University of Life Sciences. Hier beschrijven we een protocol voor te bereiden en handhaven van hypofyse en primaire celculturen van medaka (Oryzias latipes). De geoptimaliseerde voorwaarden in dit protocol rekening belangrijke parameters zoals temperatuur, osmolaliteit en pH door het nabootsen van de fysiologische omstandigheden van de vis, waardoor fysiologisch meer zinvolle resultaten.

Video: Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

Developmental Biology

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Biology

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Medicine

Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the clear majority have been performed on dissociated primary cells. To improve our understanding of how teleost pituitary cells, behave in a more biologically relevant environment, this protocol shows how to prepare viable brain-pituitary slices using the small freshwater fish medaka (Oryzias latipes). Making the brain-pituitary slices, pH and osmolality of all solutions were adjusted to values found in body fluids of freshwater fish living at 25 to 28 &#176;C. Following slice preparation, the protocol demonstrates how to conduct electrophysiological recordings using the perforated whole-cell patch-clamp technique. The patch-clamp technique is a powerful tool with unprecedented temporal resolution and sensitivity, allowing investigation of electrical properties from intact whole cells down to single ion channels. Perforated patch is unique in that it keeps the intracellular environment intact preventing regulatory elements in the cytosol from being diluted by the patch pipette electrode solution. In contrast, when performing traditional whole-cell recordings, it was observed that medaka pituitary cells quickly lose their ability to fire action potentials. Among the various perforation techniques available, this protocol demonstrates how to achieve perforation of the patched membrane using the fungicide Amphotericin B.

The article describes an optimized protocol for making viable brain-pituitary tissue slices, using the teleost fish medaka (Oryzias latipes), followed by electrophysiological recordings of pituitary cells using the patch-clamp technique with the perforated patch configuration.

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

Source: Terzi, A., et al., <a href="https://app.jove.com/v/62165">ROS Live Cell Imaging During Neuronal Development.</a> J. Vis. Exp. (2021)
The video demonstrates a method for culturing primary retinal ganglion cells from zebrafish embryos. Early-stage embryos undergo dissection to isolate the eyes, which are subsequently mechanically dissociated into a suspension of retinal ganglion cells. This cell suspension is cultured on coverslips coated with adhesive proteins to facilitate axon development.

1. Preparation of solutions

	E2 media (1x)
	
		Prepare 100x E2A (500 mL), 500x E2B (100 mL) and 500x E2C (100 mL) solutions by combining all components ...

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

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