title: Effects of pH, temperature, and osmolarity on the morphology and survival rate of primary hemocyte cultures from the mitten crab, Eriocheir sinensis.
abstract: Hemocytes are the main immune defense cells in crustacean, and its in vitro culture can be a useful tool for the study of host and pathogen interaction. In the present study, the primary hemocyte culture of Chinese mitten crab (Eriocheir sinensis), including mixed and single hemocyte, was set up for the first time. In this study, different pH (6.4, 6.8, 7.2, 7.6, and 8.0), temperature (26, 28, and 30°C), and osmolarity (500, 700, 900, 1,100, and 1,300 mOsm kg(-1)) values were tested. Moreover, the effects of two types of medium (1× L-15 and 3× L-15) with the same osmolarity on hemocyte culture were evaluated. After incubation at different culture conditions, the morphological changes (degranulation, lysis, shrinkage, and detachment) and survival rate of hemocytes were taken into account in order to evaluate the culture condition effect. Our results showed that the total hemocyte counts of Chinese mitten crab were about 2.5 × 10(7) cells ml(-1), and three subpopulations of hemocytes were distinguished as granulocytes (43.46 ± 4.98%), semigranulocytes (31.04 ± 1.95%), and hyalinocytes (25.50 ± 4.89%). The optimal culture condition for primary hemocytes of Chinese mitten crab was 3× L-15 medium, 1,100 mOsm kg(-1), pH 6.8 at 28°C. Hemocytes at optimal culture condition could retain a better morphology and higher survival rate: hemocytes retained a survival rate >60% after 5 d and >40% after 7 d. Furthermore, the hemocyte subpopulations were isolated by Percoll step gradient centrifugation and cultured in optimized hemocyte culture conditions. The results showed that hyalinocytes and semigranulocytes could maintain a survival rate of >50% after 15 d, while granulocytes only retained a survival rate of 26% after 5 d.
authors.affiliation: Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, College of Fisheries and Life Science, Shanghai Ocean University, Huchenghuan Road 999, Lingang New District, Shanghai, 201306, People's Republic of China.
authors.forename: Yuhang
authors.initials: Y
authors.lastname: Hong
authors.forename: Xiaozhen
authors.initials: X
authors.lastname: Yang
authors.forename: Yongxu
authors.initials: Y
authors.lastname: Cheng
authors.forename: Pan
authors.initials: P
authors.lastname: Liang
authors.forename: Jinbiao
authors.initials: J
authors.lastname: Zhang
authors.forename: Meng
authors.initials: M
authors.lastname: Li
authors.forename: Cheng
authors.initials: C
authors.lastname: Shen
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Yang
authors.forename: Chun
authors.initials: C
authors.lastname: Wang
mesh_terms: D000818:Animals; D003386:Brachyura / Q000166:cytology*; D018929:Cell Culture Techniques; D002470:Cell Survival; D006434:Hemocytes / Q000166:cytology*; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D013696:Temperature