The culturing techniques are important and widely used in microbiological studies. However, it has been revealed in 1980's that many microbes in the environment cannot be detected by conventional culturing techniques with long history of more than 100 years. Culturing processes are time-consuming and increase the biomass of microbial cells, which shall result to increase the risk of biohazard. We regards microbes as “particles with genetic information and physiological activity”, and develop culture-independent techniques to analyze microbial cells at the single level, a cell itself, and also community level, by using fluorescent staining methods and molecular microbial ecological approaches. By using these techniques, microbial cells can be detected and enumerated rapidly and their physiological status can be estimated at the same time. Targeted cells are detected and their dynamics can be monitored accurately based on their genetic information. Culture-independent techniques are rapid and rather simple. Results can be obtained within one hour to half a day, while conventional culturing techniques require more than a day to obtain reliable results. In order to popularize these techniques, validation and automatization is important. We applied image analysis and flow cytometry for rapid analysis. We also designed a semi-automated system for enumeration of microcolony-forming cells. Compare to conventional fluorescent microscopy, applying these new approaches is more likely to bring us more rapid and quantitative results. We now apply microfluidic devices (on-chip flow cytometry) to automatize enumeration of microbial cells, which enable us to acquire data rapidly with high reproducibility and reduce the risk of biohazard by rather simple operation. These techniques should be useful in microbiological quality assurance of freshwater.

抄録全体を表示

Regenerative medicine, which involves advanced techniques using ex vivo processed tissue-engineering products (e.g., viable cells, tissues), is one of the newest and most promising methods for treating various intractable diseases and damaged organs. For clinical use, tissue-engineered products require sterilization, so they are usually prepared manually, using aseptic processing procedures in a biological clean room (BCR) to prevent contamination with microbiological and/or hazardous materials. However, these procedures carry the unavoidable risk of contamination from the people performing them. To reduce this risk, personnel are required to wear sterilized gowns and follow written standard operating procedures (SOPs) for every process. These measures make procedures performed in the BCR laborious and expensive. In this study, to develop a system for producing tissue engineering products that is safer, less laborious, and less expensive than those used in the BCR, we examined the applicability of an isolator system for human cell cultures and developed a new system, the Advanced Isolator System for Tissue Engineering (AIST). The AIST is a compact, closed isolator system intended for aseptic human cell cultures. It has a built-in CO₂ incubator and the equipment needed for cell culture (e.g., microscope, centrifuge) is set up inside it. It is decontaminated in its entirety by vaporized hydrogen peroxide (VHP), and it can be maintained in a cleaner condition with less effort than equipment in a BCR. We have been able to culture various human cells in the microbiologically closed AIST using a half-suit unit as the operator interface, so the potential risk of contamination from personnel is as low as possible. Cell processing in the AIST will maintain the quality of human cells for clinical use at a higher level than can be attained in a BCR, and we expect it to contribute greatly to the early realization of regenerative medicine using ex vivo processed human cells.

抄録全体を表示

近年, 真空式高圧蒸気滅菌器の管理にBowie-Dickテストが用いられるようになった。英国基準であるHTM2010はBowie-Dickテストが不合格になったときには真空リークテストを行うことを推奨している。真空リークテストを用いるとBowie-Dickテストが不合格になるおもな機械的原因である真空ポンプの機能低下と缶体内への空気漏れの有無を評価できる。ところが, 当手術部に設置された真空式高圧蒸気滅菌器には真空リークテストを行うための自動テスト機能が組み込まれていない。そこで, 真空リークテストを用手的操作を用いて行う方法を開発し, その有用性を検討した。真空リークテストはHTM2010にしたがって行った。対象とした滅菌器においてBowie-Dickテストが不合格になったときに真空リークテストを用手的操作を用いて行った。真空ポンプの機能を示す真空度は164.3mbar absolute, 真空リーク速度は0.10mbar/minであった。HTM2010では真空度は70mbar absolute以下, 真空リーク速度は1.3mbar/min以下であると規定しているので, 真空ポンプの機能低下が疑われた。真空ポンプを交換したところ, Bowie-Dickテストは合格した。さらに, 再施行した真空リークテストもHTM2010の規定を満たした。また, 機能低下した真空ポンプを分解したところ, 錆びによる腐食劣化が広範に認められた。腐食劣化の原因は現在, 調査中である。われわれが開発した, 真空リークテストを用手的操作を用いて行う方法は有用であるとともに, Bowie-Dickテストが不合格になった真空式高圧蒸気滅菌器の修理において有用であることが示唆された。

抄録全体を表示