Thomas B. Shea

A serum-free medium that supports the growth of piscine cell cultures

SummaryAn undefined, serum-free medium was developed for use with fish cell cultures. Lactalbumin hydrolyzate, trypticase-soy broth, Bacto-peptone, dextrose, yeastolate, and polyvinylpyrrolidone were initially combined in 100 ml of distilled H2O, autoclaved, and added to 5% of the final volume of Medium 199. In addition, filter sterilized bovine pancreatic insulin, glutamine, and nonessential amino acids were added to the medium. The addition of insulin was observed to be unnecessary. Five fish cell lines [goldfish-derived CAR cells, fathead minnow (FHM) cells, epithelioma papillosum cyprini (EPC) cells, chinook salmon embryo (CHSE-214) cells, and a new cell line from goldfish air bladders (ABIII)] were all capable of growth in the serum-free medium at rates equivalent to cells grown in fetal bovine serum (FBS). The morphology of all cell lines, except CHSE-214 cells, was identical to cells grown in FBS. All cell lines were capable of long-term growth in the serum-free medium. The CAR, ABIII, EPC, and CHSE-214 cells in the serum-free medium supported the replication of goldfish virus-2 at levels equivalent to cells grown in FBS.

A simplified method for in situ embedding of monolayer cultures for electron microscopy

A simple method for the in situ embedding of cell cultures for electron microscopy has been developed. The procedure is carried out in its entirety within the culture flask, using only standard fixing and embedding reagents. Precoating of the supporting surface of the flask with alternative substrates is not required. This method permits precise orientation of samples with respect to the phase-contrast image of living cultures.

A simple method for obtaining cultures enriched for Type II pneumonocytes from fetal rabbit

A differential plating method permitted preparation of cultures significantly enriched for Type II pneumocytes. These cells were maintained in a differentiated state for at least 12 d, identifiable morphologically (by presence of osmiophilic lamellar inclusion bodies) and bio-chemically (by demonstration of synthesis of phosphatidyl choline and production of disaturated lecithin).

Chronic exposure of goldfish-derived cell cultures to toxaphene alters the replication of Goldfish Virus-2

In a recent examination of the effect of toxaphene on the goldfish-derived CAR cell line, it was demonstrated that cells chronically exposed to low levels of toxaphene were ~ore sensitive to subsequent exposure to increased concentrations of this insecticide than were cells which had not been previously exposed (SHEA & BERRY 1982). To further explore this phenomenon, we examined the response of chronically exposed cells to infection by Goldfish Virus-2. MATERIALS & METHODS Cells and virus: cultivation of the goldfish-derived CAR cell line (ATCC CCL-71) has been previously described (SHEA & BERRY 1982). Goldfish Virus-2 (GFV-2), an Iridovirus, was originally isolated from air bladder cultures of healthy goldfish in this laboratory (BERRY & SHEA 1982); viral growth studies have indicated GFV-2 replication is completed by 5 days in CAR cell cultures at 25~ Medium: Medium 199 was employed, supplemented with 10% fetal bovine serum, and I00 units of penicillin and streptomycin and 0.025 mcg. Fungizone/mL (Gibco). Toxaphene: technical grade toxaphene (99.5% pure) was obtained from Hercules Corp., Delaware. Stock solutions were prepared in 100% ethanol at 0.2g/mL and serially diluted with medium to final concentrations of 50, 35, 25, 15, I0~ and 5 parts per million (ppm). Chronic exposure of cells: cells were chronically exposed to toxaphene at 5ppm for I00 days as previously described (SHEA & BERRY 1982). In brief, cells were planted in 150cm 2 flasks (Corning) at a density of 2xl06/flask in 20mL medium without toxaphene and incubated for 24hr at 25~ Medium was then replaced with 20mL medium containing 5ppm toxaphene. At 10-day intervals, cells were subcultured, with the above proceedure repeated. Infection of chronically-exposed cells: after i00 days chronic exposure to 5ppm toxaphene (above), cells were planted in 24-well trays (Falcon) at a density of ix105/well in medium without toxaphene; these cells were termed CAR 5. Control cells (CARo) were passaged in identical manner in medium without toxaphene. 24hr later, medium was removed, and duplicate cultures received 2xlO6TCID50 GFV-2. After allowing 2hr for viral adsorption, cultures were rinsed and refed with medium without toxaphene. The viral inoculum was retained for titration of unadsorbed virus. Viral progeny were harvested on day 5 after infection. TCID50 were determined by endpoint dilution using the microtitration system (BERRY & SHEA 1982). Total protein/culture was determined according to OYAMA & EAGLE (1956).

Air bladder cells: A new continuous cell line from goldfish

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