Hiroyuki Ohno1, Akiyuki Murano. 1. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan. hiroohno-cib@umin.ac.jp
Abstract
AIM: To perform a long culture passage of H pylori without serum, taking into account its cytotoxicity and the presence of the probable new cytotoxic factor. METHODS: One sample of H pylori 60190 (ATCC 49503) was grown on Brain Heart Infusion (BHI) agar containing 0.5% 2,6-di-O-methyl-beta-cyclodextrin without any serum, being passaged 70-100 times every 3-4 d for approximately 2 h, while another sample of H pylori contained 70 mL/L fetal calf serum without 2,6-di-O-methyl-beta-cyclodextrin. Their supernatant and extract after 16 h in culture were evaluated for changes in cell morphology and for cell viability using HeLa cells. Furthermore, the characteristics of the probable cytotoxic factor in the extract were examined on partial purification studies and its cytotoxicity was evaluated in various human cells. RESULTS: The supernatant and the extract of the bacterium grown on serum-free medium had strong cytotoxicity compared with those grown on serum-containing medium. They irreversibly damaged HeLa cells without vacuolation that was altogether different from that of the bacterium when grown with serum. Their cytotoxicity was easily measured by cell viability assay. The probable cytotoxic factor partially purified and detected by chromatography had characteristics difference from that of vacuolating toxin and a broad cytotoxicity toward various cell lines. CONCLUSION: Serum-free long culture method of H pylori makes its supernatant and its extract cytotoxic enough to be easily measured by cell viability assay. The probable cytotoxic factor has a unique characteristic and might be a new cytotoxin.
AIM: To perform a long culture passage of H pylori without serum, taking into account its cytotoxicity and the presence of the probable new cytotoxic factor. METHODS: One sample of H pylori 60190 (ATCC 49503) was grown on Brain Heart Infusion (BHI) agar containing 0.5% 2,6-di-O-methyl-beta-cyclodextrin without any serum, being passaged 70-100 times every 3-4 d for approximately 2 h, while another sample of H pylori contained 70 mL/L fetal calf serum without 2,6-di-O-methyl-beta-cyclodextrin. Their supernatant and extract after 16 h in culture were evaluated for changes in cell morphology and for cell viability using HeLa cells. Furthermore, the characteristics of the probable cytotoxic factor in the extract were examined on partial purification studies and its cytotoxicity was evaluated in various human cells. RESULTS: The supernatant and the extract of the bacterium grown on serum-free medium had strong cytotoxicity compared with those grown on serum-containing medium. They irreversibly damaged HeLa cells without vacuolation that was altogether different from that of the bacterium when grown with serum. Their cytotoxicity was easily measured by cell viability assay. The probable cytotoxic factor partially purified and detected by chromatography had characteristics difference from that of vacuolating toxin and a broad cytotoxicity toward various cell lines. CONCLUSION: Serum-free long culture method of H pylori makes its supernatant and its extract cytotoxic enough to be easily measured by cell viability assay. The probable cytotoxic factor has a unique characteristic and might be a new cytotoxin.