ISSN:
1573-5117
Keywords:
cell culture
;
density gradients
;
Dugesia polychroa
;
neoblast separation
;
planarians
;
Tricladida
;
Turbellaria
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract For highly purified preparations of neoblasts, density gradient centrifugation in Percoll solutions (Pertoft et al., 1978) was applied to cell suspensions obtained by disintegrating Dugesia polychroa (Schmidt) in culture medium contained in a Dounce homogenizer (tolerance: 50 µm; one animal 12 mm in length per ml). To reduce the high viscosity caused by mucus, 0.00063% (w/v) of dithiothreitol was added during disintegration and purification. Based on previous experiments (Schürmann & Peter, 1988), five media were compared. For prepurification, four washing steps (differential centrifugations at 500 × g for 5 min each) were followed by subsequent filtration through a series of nylon gauzes (40, 30, 20 and 15 µm mesh size) and a final washing step. The resulting cell suspensions were then fractionated by isopycnic centrifugation (500 × g, 45 min) in one continuous (1.018–1.121 g ml−1) or one of seven different discontinuous Percoll gradients (Schürmann, 1993). The best yield and highest purity of neoblasts in one fraction was obtained with a four step gradient (1.03–1.09 g ml−1): the neoblasts (purity: 91%) were concentrated in one sharp band at the boundary between the densities 1.05 and 1.07 g ml−1. The spherical cells (diameter from 10 to 13 µm in vivo) stained as typical neoblasts (Pedersen, 1959). Primary cultures were obtained with all media. The medium developed by Teshirogi and Tohya (1988) and its isotonic modification (Schürmann, 1993) proved best, resulting in 86% of viable cells without signs of differentiation after 17 days of culture at 18 °C, with still 46% being left after 31 days. Earlier reports state that isolated neoblasts only survive for 4 days (Betchaku, 1967) and total planarian cell suspensions only 2–3 weeks (Teshirogi & Tohya, 1988).
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00036403
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