Abstract:

Objective To establish a more effective and economical method for primary culture of germinal cells released from Echinococcus granulosus hydatid cyst walls. Methods The cyst walls of E. granulosus hydatids isolated from the abdominal cavities of infected mice were digested with 0.25% trypsin-EDTA solution for 10, 20 and 30 minutes, respectively, the digested cyst walls debris were then primarily cultured using the regular debris-adherent culture method. The undigested cyst walls were cultured using the same adherent method as control. The growth of germinal cells was observed under an inverted microscope for their dynamic features of cell morphology and growth curve. The germinal cells were recognized by the serum from a patient with hydatidosis using immunofluorescence assay. To confirm the successful generation of E. gramulous germinal cells, the cultured cells were used to inoculate naive mice to observe the generation of hydatids 4 months after inoculation. Results The germinal cells in the cyst wall debris digested for 10 minutes were intact and full of cytoplasm. Plenty of germinal cells were grown and outreached from the edges of debris 2-4 days after being cultured and the colony-like growth of germinal cells was found 9 days post culture. However, the germinal cells in the cyst wall debris digested for 20 minutes or 30 minutes were mostly broken with naked nuclei observed. The success rate for the generation of germinal cells from the digestion-adherent culture method with digestion for 10 minutes (6/10) was higher than that generated from the direct adherent culture method (3/10). The growth curves of both methods were similar, with a “S” growth pattern. The average culture period for the generation of germinal cells from the hydatid cyst walls using the digestion culture method was 18 days. The proliferation level of germinal cells released from the digested cyst walls debris was higher than that released from the direct adherent culture (P < 0.01). The generated germinal cells from the digested culture for 18 days could be recognized by the serum from a patient with hydatidosis. The germinal cells collected from the digested culture were able to infect normal mice with cystic vesicles generated in the abdominal cavities of the mice inoculated with the culture cells in 4 months. HE staining of the cyst sections showed that the generated cysts contained the developing laminated layer and the germinal layer observed under microscope. Conclusion The modified adherent culture method of cyst wall with 0.25% trypsin-EDTA digestion for 10 minutes generated higher rate of germinal cells with ability to infect mouse.

Key words: Echinococcus granulosus, Germinal cell, Debris, Cell culture, Inoculation