ABSTRACT
OBJECTIVE: To develop a reliable method to measure host cell protein(HCP)content of HEK293 cells in rAAV2-KAL vectors for clinic application. METHODS: The rAAV2-KAL vectors used for this study was purified by CsCl ultracentrifugation twice,then were subjected to by Western blot. The optimal linear range, minimum detection limit,and the accuracy and precision were verified by ELISA. Three batches of rAAV2-KAL were prepared by CsCl method. The change of HCP content during purification was determined to verify the method suitability and reliabilities. RESULTS: The optimal linear range of the method developed was 4-200 ng·mL-1, while the minimum detection limit of 4 ng·mL-1. The recovery rates of HEK293 cell protein at various concentrations were at range of 77.0%-115.0%,with a coefficient of variation of less than 20%. The HCP contents in three batches of rAAV2-KAL were less than 100 ng·mL-1, all data taken together indicated that HCP was effectively removed by CsCl ultracentrifugation. CONCLUSION: A reliable ELISA assay for residual host cell protein of HEK293 cells is successfully developed,which might be used for determination of HCP content in CsCl ultracentrifugation purified rAAV2-KAL for clinical application.
ABSTRACT
The biological characterization, differentiation and regeneration of hepatic stem/progenitor cells are the one of very active and interested fields. In this report, intravenous injection of human umbilical cord blood (HUCB) cells into the BALB/c-nu and SCID mice, an animal model for transplantation and liver injury, was reported. Using of flow cytometry and tissue typing (HLA), it was found that the HUCB cells were survived in mouse liver for 9 weeks. After separation from perfused liver, HUCB cells were detected by hematopoietic colonies (CFU-GEM M) in hepatocyte culture. It was concluded that the transplanted HUCB hematopoietic stem/progenitor cells can be survived in the liver over a long period of time.