Chinese Shrimp Cell Culture Research And Its Applications

Chinese shrimp, Fenneropenaeus chinensis, is one of the most commerciallyimportant invertebrates cultured in China. The present paper reports in vitro culture ofthe haemocytes and the cells of embryo, including blastula and gastrula, from theshrimp. Haemocytes of crustacean play an important and central role in host defense.It is widely recognized that embryonic cells are undifferentiated and can rapidlymultiply. Especially, the cell cultures of early developing embryo, including morulaand blastula, are one of two sources of embryonic stem(ES)cells. Furthermore, invitro infection of the primary adherently cultured haemocytes of the shrimp withwhite spot syndrome virus(WSSV) was carried out, and the transient expression offoreign genes in the primary cell cultures of the shrimp was observed. The presentwork is to facilitate the study of shrimp immunology and transgenic studies bydeveloping a primary culture system of the shrimp. Some important progresses were ii余黎明 中国对虾细胞培养研究及其初步应用 硕士学位论文abstained: 1. The primary culture of the peripheral haemocytes from the shrimp was carriedout. The effects of different medium and method of the collection of the haemocytesfrom the shrimp on their primary adherent culture were observed. The optimalmedium(L-15＋10%BCS＋20% or 25%ME, pH 7.0~7.2)was based on 1×L-15medium supplemented with 10% bovine calf serum, 20% or 25% muscle extract fromthe shrimp , F. chinensis, and also 1.0g/L glucose , 12.0g/L NaCl(used to regulate theosmolarity) and antibiotics. A monolayer culture in vitro that were sub-confluent fora minimum of 8 days has been established from peripheral haemocytes of the shrimpwith the optimal medium at the temperature of 23.0℃. Two kinds of anticoagulantswere suitable for collecting the haemocytes. They were both pH 7.0 aqueous solution,one contained 450mmol/L NaCl, 10mmol/L KCl, 10mmol/L EDTA． Na2 and10mmol/L HEPES, the other contained 336mmol/L NaCl, 115mmol/L Glucose,27mmol/L Na3C6H5O7 and 9mmol/L EDTA．Na2. It is a key for the culture to sterilizefully and to keep the viability of the haemocytes simultaneously, and sterilizing in500mg/L iodophore solution after anaesthetizing with 0~4℃ fresh water could meetthis need. The plasma of the shrimp were deleterious to the primary culturedhaemocytes in vitro, and replacing all media and plasma with new media after thehaemocytes' adhering could avoid the deleterious effect. Furthermore, the haemocytessuspension culture and serum-free culture were tried, and the above-mentionedmedium, L-15＋10%BCS＋20%ME, was suitable for the former, while the modifiedPBS was suitable for the latter. 2. In vitro cell culture of the embryo, including the blastula and gastrula, of F.chinensis was carried out. Viable blastula cells were obtained by dissecting the eggs(puncturing the chorion with fine acus and then gently pipetting the blastula cellmass), and good yields of viable gastrula cells were obtained by crushing the eggsgently. The attachment and spread of the embryonic cells could remarkably improvedby decreasing the concentration of fetal bovine serum(FBS) and the volume of theculture system at the beginning of the culture. After spreading, blastula cells took ongrowth hallow, but they couldn't proliferate notably, while the cells of the gastrula iii余黎明 中国对虾细胞培养研究及其初步应用 硕士学位论文developed for 13~18h could easily become sub-confluent. However, passage of theprimary cultures resulted in the loss of adherence of the cells. The medium used was 1×Leibovitz's L-15 medium supplemented with 12.0g/L NaCl, FBS and embryoextract of F. chinensis , and the concentration of FBS and the embryo extract wasbased on the need of different experiments. It was worth the whistle that the blastu...