Purchase this article with an account.
Yoko Oumi, Masukazu Inoie, Miho Fujita, Yuko Suwa, Takahiro Ogasawara, Chikara Shinohara, Ken-ichiro Hata; Quantitative Evaluation Of Bovine Serum, Cholera Toxin, And Antibiotics Remaining In Human-cultured-limbal Epithelium Based On Addition-recovery Tests. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1949.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Autologous human-cultured-limbal epithelium (HCLE) manufactured by using fibrin glue and 3T3-J2 cells as feeder layer have good effect for treatment of limbal stem cell deficiency. We have been developing the cellular product of autologous cultured limbal epithelium in Japan. According to Japanese Pharmaceutical Affairs Act, medicinal products such as cell based materials must be evaluated the residual level of the additive supplements contained in culture medium for safety control. Fetal bovine serum (FBS), cholera toxin (CT), and antibiotics (Abs) were used in the manufacturing processes of HCLE. In this study, we have evaluated the each amount of remaining FBS, CT, and ABs in it, using original appropriate quantitative method based on addition-recovery tests.
Limbal epithelial cells were isolated by trypsinization from human cornea and inoculated with X-irradiated 3T3-J2 feeder layer on culture flask. After cell cultivation at 37°C, 5% CO2, limbal epithelial cells, almost confluent, were subcultured on fibrin glue to produce HCLE. For measurement of each value of HCLE, the lysate were determined by using ELISA-kit of bovine serum albumin for FBS, original sandwich-ELISA method for CT, and the liquid chromatography tandem mass spectrometry (LC-MS/MS) for Abs, respectively. Each method for quantitative determination was preliminarily validated by appropriate addition-recovery test.
The mean amount of BSA remained in HCLE was 17 µg per sheet. The each remained amount of CT and all ABs was less than the limit of detection.
Quantity of residual FBS, CT and ABs in HCLE should be determined for product safety under Japanese Pharmaceutical Affairs Act. FBS was measured as BSA amount because protein mixture. CT was not able to be measured accurately by normal ELISA, thus we had developed the original sandwich-ELISA technique. In determination of ABs, the minimum detectable quantity had been downed by LC-MS/MS. It is very important to construct the reliable measurement methods to ensure the safety of cellular product.
This PDF is available to Subscribers Only