Document Type : Research Paper

Authors

1 1 Student Research Committee, Babol University of Medical Sciences, Babol, I.R. Iran. 2 Biomedical and Microbial Advanced Technologies (BMAT) Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, I.R. Iran.

2 1. Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, I.R. Iran. 2. Biomedical and Microbial Advanced Technologies (BMAT) Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, I.R. Iran.

3 Immunology, Asthma and Allergy Research Institute, Children’s Medical Center Hospital, Tehran University of Medical Sciences. Tehran, Iran

4 1. Immunology, Asthma & Allergy Research Institute (IAARI), Tehran University of Medical Sciences, Tehran, Iran. 2. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.

10.30498/ijb.2024.409915.3772

Abstract

Abstract

Background: Coagulation factor VIII (FVIII) is applied for spontaneous hemorrhaging inhibition and excessive bleeding after trauma in patients with hemophilia A. High-quality human recombinant factor VIII (rFVIII) has been produced relatively in large quantities in cultured mammalian cells. NS0 is one of the most common mammalian cell lines for therapeutic protein production. Production of rFVIII has increased due to low FVIII expression levels and rising demand for hemophilia A prophylactic treatment. Several methods have been developed to prevent cell cycle progression in mammalian cells for increased recombinant protein yields.

Objective: The aim of the study was to investigate the level of recombinant BDD-FVIII expression in NS0 mouse myeloma cells. Additionally, the study aimed to determine the effects of chemical drugs, Mitomycin C, Lovastatin, and Metformin on the secretion of FVIII through cell cycle arrest.

Materials and Methods: We cultured NS0 cells and transfected them with the 2 μg pcDNA3-hBDDFVIII plasmid by Lipofectamine 3000. The cells were treated with 10 μg.ml^-1 Mitomycin C, 20 μM Lovastatin, and 20 mM Metformin separately. After 24 and 48 hours, the samples were collected and, protein expression was analyzed using RT-PCR, Dot blot, and ELISA.

Results: A higher protein expression level was observed in treated cells 24h and 48h after treatment with all three drugs. According to real-time PCR, Metformin treatment resulted in the highest expression level within 24 h (P=0.0026), followed by Mitomycin C treatment within 48 h (P=0.0030).

Conclusion: The NS0 cell line can be regarded as a suitable host for FVIII production. FVIII protein expression level was increased by using Lovastatin, Metformin, and Mitomycin C drugs. Further investigations are suggested, and the potential application of these drugs to increase recombinant protein yield can be used to produce therapeutic proteins in the industry.

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