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

Summary

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.

Abstract

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.

Introduction

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 (3–4 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 4–40 °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 °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 275–295 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 °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 °C to pH 7.75 for medaka kept at 26 °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.

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Protocol

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

1. Calibrate the osmometer and pH meter instruments according to the manufacturer’s instructions to ensure correct measurements.
2. Prepare 500 mL of Ca²⁺- and Mg²⁺-free Dulbecco’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).
   1. 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.
   2. 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.
   3. Sterile-filter the dPBS solution using a 0.2 µm filter.
      NOTE: The solution may be stored at 4 °C for at least 3 months.
3. 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 °C for at least 4 weeks.
   1. Add 420 mg of NaHCO₃ per 500 mL of culture medium to achieve a 10 mM solution.
   2. Add 405 mg of glucose per 500 mL of culture medium to achieve a 4.5 mM solution.
   3. 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.
   4. Adjust the solution to 290 mOsm/kg with mannitol using an osmometer (as performed in step 1.2.2).
   5. Perform a sterile filtration of the L-15 medium using a 0.2 µm filter.
   6. After the sterile filtration, add 2.5 mL of a penicillin-streptomycin solution, corresponding to 50 U/mL of penicillin and 50 µg/mL of streptomycin.
4. 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 °C until use.
   NOTE: Aliquots of 2 mL can be stored at -20 °C for at least 3 months.
5. 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 µg/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 °C until use.
   Note: Aliquots of 2 mL can be stored at -20 °C for at least 3 months.

2. Preparation of Equipment

1. Prepare glass pipettes by fire polishing the tip for the cell dissociation. Make tips with 2 different size categories (a medium opening of 0.6–0.8 µm and a small opening of 0.4–0.6 µm) 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 °C for 45 min).
2. Prepare a polystyrene box with ice.
3. 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.
4. 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.
5. 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.
6. Set the water bath to 26 °C before starting the dissection to allow the water to reach the desired temperature before the chemical dissociation of the pituitaries.
7. 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.

3. Pituitary Dissection and Cell Dissociation

1. Euthanize the fish by immersing it in ice slush (0 °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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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 2–3 min per fish, of which only around 10 s are needed to pick the pituitary when the brain is already exposed.
7. 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. 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).
8. Spin down the pituitaries in a tabletop centrifuge for 1–2 s at room temperature. Carefully remove the dPBS using a glass pipette and take care to avoid removing any pituitaries.
9. Add 1 mL of the trypsin solution (prepared in step 1.4) to wash the pituitaries, spin them down in a tabletop centrifuge for 1–2 s, and remove most of the liquid using a glass pipette before adding an additional 1 mL of trypsin solution.
10. Incubate the tube in a water bath at 26 °C for 30 min, preferably with gentle shaking, or alternatively, flick the tube a couple of times during the incubation period.
11. Spin down the pituitaries in a tabletop centrifuge for 1–2 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.
12. Collect the pituitaries at the bottom of the tube by spinning them down in a tabletop centrifuge for 1–2 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 °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.
13. 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 °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).
14. Set the cooling centrifuge for the 15 mL tubes to 4 °C to allow it to reach the desired temperature before starting the mechanical dissociation.
15. 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 1–2 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.
16. 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 (6–7x) in a fire-polished glass pipette (prepared in step 2.1).
    NOTE: Temperature is an essential aspect, so keep all solutions at 4 °C. From this point onward, perform all steps on ice when possible.
17. Spin down the tissue pieces in a tabletop centrifuge for 1–2 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.
18. Repeat step 3.16–3.17, 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 3–4 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.
19. Balance the tube in the centrifuge (pre-cooled to 4 °C) and spin down the cells at 200 x g for 10 min at 4 °C. Make sure to mark the side of the tube where the cell pellet will be.
20. 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.
21. Carefully resuspend the cell pellet in 50–100 µL 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.
22. 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.
23. Incubate the dish at 26 °C and 1% CO₂ to allow all the cells to sink thoroughly and attach to the bottom of the dish.
24. After 30 min, look at the cells in the microscope to make sure they have attached to the culture dish.
25. Continue to incubate the cells at 26 °C and 1% CO₂.
26. Carefully change most of (but not all) the medium every 3–4 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.

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Results

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 (

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Discussion

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...

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Materials

Name	Company	Catalog Number	Comments
Dulbecco’s Phosphate Buffered Saline (dPBS), without calcium chloride and magnesium chloride	Sigma (Merck, Damstadt, Germany)	D8537	Adjust solution to pH 7.75 with 1M NaOH and 290 mOsm with mannitol.
L-15 medium (Leibovitz), witout L-glutamine	Sigma (Merck, Damstadt, Germany)	L5520	Supplement 500 ml culture medium with 10 mM NaHCO3, 4.5 mM glucose, 2 mM Glutamax. Adjust solution to 290 mOsm with mannitol and filter solution through a 0.2 µm PES sterile filter, before adding 2.5 mL Penicillin-Streptomycin solution (see below for details).
NaOH	Sigma (Merck, Damstadt, Germany)	S5881	Add drops of 1 M solution to increase pH of dPBS and culture medium to 7.75.
NaHCO3	Sigma (Merck, Damstadt, Germany)	S5761	10 mM NaHCO3 equals 420 mg per 500 mL culture medium.
D-mannitol	Sigma (Merck, Damstadt, Germany)	63565	Use to increase osmolality of dPBS and culture medium. Calculate correct amount needed to reach an osmolality of 290 mOsm.
D-glucose	Sigma (Merck, Damstadt, Germany)	G5400	4.5 mM  D-glucose equals 405 mg per 500 mL culture medium.
GlutaMAX Supplement	Gibco (Life Technologies, Paisley, UK)	35050-061	Alternative to L-glutamine, with increased stability. 2 mM Glutamax equals 5 ml of 100x stock in 500 mL culture medium.
Penicillin-Streptomycin	Sigma (Merck, Damstadt, Germany)	P0781	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 µg/mL Streptomycin).
Trypsin type II-S	Sigma (Merck, Damstadt, Germany)	T7409	Prepare 1 mg/mL in dPBS solution.
Trypsin inhibitor type I-S	Sigma (Merck, Damstadt, Germany)	T6522	Prepare 1 mg/mL in dPBS solution, supplement with 2 µg/ml Dnase I (see details below).
Dnase I	Sigma (Merck, Damstadt, Germany)	D5025	Use in trypsin inhibitor solution (see above).
0.2 µm Polyethersulfone (PES) sterile filter system	Corning Inc. (Corning, NY)	431097	Use for sterile filtration of dPBS and L-15 medium after adjustments.
35 mm cell culture dish with glass bottom, poly d-lysine coated	MatTek Corporation (Ashland, MA)	P35GC-1.5-10-C	Can also be replaced by plastic dish, depending on downstream application.
Dumont #5 fine forceps	Fine Science Tools (CA)	11254-20	Straight tip
Dumont #5/45 fine forceps	Fine Science Tools (CA)	11253-25	Angled 45° tip
Stereo microscope SZ61	Olympus Corp. (Tokyo, Japan)		Use for dissection of pituitaries.
Alegra X-22R Centrufuge	Beckman Coulter Inc. (Brea, CA)		With cooling option (similar to current model XR-30).
Water bath	Techne (Staffordshire, UK)		Any water bath with the possibility of adjusting temperature will do.
Pasteur glass pipettes	VWR (NY)	612-1701	Outer diameter 1.6 mm, fire polish and autoclave before use.
Galaxy MiniStar table centrifuge	VWR (NY)	521-2844	Any small table centrigue will do.
Fine needles/insect pins	Fine Science Tools (CA)	26001-40	Diameter 0.03 mm. Other fine needles can be used instead.
Wax plate			Custom made by adding melted paraffin wax in large petri dish.