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

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.

Gnrh1-induced responses are indirect in female medaka Fsh cells

“…During the initial current clamp recordings, we observed spontaneous firing of action potentials in 60% (n = 20) of the Fsh cells (Figure 4A), similar to our previous results on medaka Lh cells (21,22,43). The spontaneous firing of Fsh cells occurred in cells with oscillating membrane potential around −45 to −40 mV with the action potential often overshooting 0 mV (liquid junction potential not corrected for).…”

“…(B) Typical current clamp recording of a spontaneously firing Lh cell. Gnrh1 application using puff ejections induced a biphasic response with an initial hyperpolarization followed by depolarization and increased firing and/or increase action potential duration (see also (43)). (C) Ca 2+ imaging of an Fsh cell.…”

“…Pituitaries were collected from 10 mature, gravid females from the tg( lhb :hrGfpII) and tg( fshb :DsRed2) lines separately following decapitation and dissociated according to (43). In brief, pooled pituitaries were digested with trypsin (2 mg/mL, Sigma-Aldrich) for 30 min at 26°C, then incubated with trypsin inhibitor (1 mg/mL, Sigma-Aldrich) and DNase I Type IV (2 μg/mL, Sigma-Aldrich) for 20 min at 26°C with gentle shaking.…”

“…All electrophysiological experiments were conducted using the perforated patch clamp technique in current clamp on either brain-pituitary slices (44) or primary dissociated cells (43) from the tg lines described above ( Live brain-pituitary slices ), and with ECS containing 2 mM Ca 2+ and 0.1% BSA. The experiments were conducted between 14:00 h and 19:00 h. Patch pipettes were made from thick-walled borosilicate glass with 4-5 MΩ resistance.…”

Gonadotropes in medaka grow long extensions with varicosity-like swellings, projecting towards each other and blood vessels

“…The cell cultures were made as previously described (Ager-Wick et al, 2018;Strandabo et al, 2013). In brief, the fish were anesthetized, then the pituitary was dissected out and enzymatically digested with trypsin (2 mg/mL, Sigma-Aldrich) for 30 min at 26 ºC, followed by incubation with trypsin inhibitor (1 mg/mL, Sigma-Aldrich) and Dnase I Type IV (2 μg/mL, Sigma-Aldrich) for 20 min at 26 ºC with gentle shaking.…”