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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

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

Published: August 28th, 2018

DOI:

10.3791/58159

1Department of Basic Sciences and Aquatic Medicine, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 2Department of Oral Biology, Faculty of Dentistry, University of Oslo

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 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.

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....

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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 7.7.......

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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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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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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.

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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 10mM 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 1M 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 Straigt 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.

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