The production of an Fab fragment of the catalytic antibody 6D9 in stably transformed lepidopteran insect cells was investigated. On the basis of an expression vector that utilizes the Bombyx mori cytoplasmic actin promoter, from which foreign gene expression is stimulated with the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer, two plasmid vectors were constructed which contain either a neomycin or a blasticidin resistance gene for use as a selectable marker. The genes encoding the heavy chain (Hc; Fd fragment) and light chain (Lc) of the 6D9 Fab fragment were inserted separately into the expression vectors. After cotransfection with the resulting plasmids to introduce the Hc and Lc genes and the two different antibiotic resistance genes, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were cultured in the presence of G418 and blasticidin. Colonies of cells resistant to the antibiotics were obtained around 2 weeks after cotransfection. Western blotting and enzyme-linked immunosorbent assay (ELISA) of the cell culture supernatant suggested that the resistant cells stably secrete an Fab fragment which retains an antigen-binding activity. High yields of over 300 μg/ml of Fab fragment were achieved in simple batch shake-flask culture of transfected insect cells. These results indicate that recombinant insect cells may offer a novel approach for efficient production of antibody molecules.
BackgroundCell-based regeneration therapies have great potential for application in new areas in clinical medicine, although some obstacles still remain to be overcome for a wide range of clinical applications. One major impediment is the difficulty in large-scale production of cells of interest with reproducibility. Current protocols of cell therapy require a time-consuming and laborious manual process. To solve this problem, we focused on the robotics of an automated and high-throughput cell culture system. Automated robotic cultivation of stem or progenitor cells in clinical trials has not been reported till date. The system AutoCulture® used in this study can automatically replace the culture medium, centrifuge cells, split cells, and take photographs for morphological assessment. We examined the feasibility of this system in a clinical setting.ResultsWe observed similar characteristics by both the culture methods in terms of the growth rate, gene expression profile, cell surface profile by fluorescence-activated cell sorting, surface glycan profile, and genomic DNA stability. These results indicate that AutoCulture® is a feasible method for the cultivation of human cells for regenerative medicine.ConclusionsAn automated cell-processing machine will play important roles in cell therapy and have widespread use from application in multicenter trials to provision of off-the-shelf cell products.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.