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Background: Chinese hamster ovary (CHO) cells are the most predominantly utilized host for the production of monoclonal antibodies (mAbs) and other complex glycoproteins. A major challenge in the process of CHO cell culture is the occurrence of cell death following different stressful conditions, which hinders the production yield. Engineering genes involved in pathways related to cell death is a remarkable strategy to delay apoptosis, improve cell viability and enhance productivity. SIRT6 is a stress-responsive protein that regulates DNA repair, maintains genome integrity, and is critical for longevity and cell survival in organisms. Methods and results: In this study, SIRT6 was stably overexpressed in CHO-K1 cells and the impact of its expression on apoptosis related gene expression profile, viability, apoptosis, and mAb productivity was investigated. While a significant increase was observed in Bcl-2 mRNA level, caspase-3 and Bax mRNA levels were decreased in the SIRT6 engineered cells compared to the parental CHO-K1 cells. Moreover, improved cell viability and decreased rate of apoptotic progression was observed in a SIRT6-derived clone in comparision to the CHO-K1 cells during 5 days of batch culture. anti-CD52 IgG1 mAb titers were improved up to 1.7- and 2.8-fold in SIRT6-derived clone during transient and stable expression, respectively. Conclusions: This study indicates the positive effects of SIRT6 overexpression on cell viability and anti-CD52 IgG1 mAb expression in CHO-K1 cells. Further studies are needed to examine the potential of SIRT6-engineered host cells for the production of recombinant biotherapeutics in industrial settings. [ABSTRACT FROM AUTHOR]

Fibroblasts are the main cells of connective tissue and have pivotal roles in the proliferative and maturation phases of wound healing. These cells can secrete various cytokines, growth factors, and collagen. Vascular endothelial growth factor (VEGF) is a unique factor in the migration process of fibroblast cells through induces wound healing cascade components such as angiogenesis, collagen deposition, and epithelialization. This study aimed to create VEGF165 overexpressing fibroblast cells to evaluate angiogenesis function in wound healing. In vitro, a novel recombinant expression vector, pcDNA3.1(-)-VEGF, was produced and transfected into the fibroblast cells. Following selecting fibroblast cells with hygromycin, recombinant cells were investigated in terms of VEGF expression by quantifying and qualifying methods. Mechanical, physical, and survival properties of polyurethane-cellulose acetate (PU-CA) scaffold were investigated. Finally, in vivo, the angiogenic potential was evaluated in four groups containing control, PU-CA, PU-CA with fibroblast cells, and VEGF-expressing cells on days 0, 2, 5, 12 and 15. Wound biopsies were harvested and the healing process was histopathologically evaluated on different days. qRT-PCR showed VEGF overexpression (sevenfold) in genetically-manipulated cells compared to fibroblast cells. Recombinant VEGF expression was also confirmed by western blotting. Manipulated fibroblast cells represented more angiogenesis than other groups on the second day after surgery, which was also confirmed by the antiCD31 antibody. The percentage of wound closure area on day 5 in genetically-manipulated Hu02 and Hu02 groups showed a significant reduction of wound area compared to other groups. These findings indicate that overexpression of VEGF165 in fibroblast cells results in enhanced angiogenesis and formation of granulated tissue in the early stage of the healing process, which can show its therapeutic potential in patients with impaired wound healing and also provide functional support for gene therapy. [ABSTRACT FROM AUTHOR]

Wound healing is a serious obstacle due to the complexity of evaluation and management. While novel approaches to promoting chronic wound healing are of critical interest at the moment, several studies have demonstrated that combination therapy is critical for the treatment of a variety of diseases, particularly chronic wounds. Among the various approaches that have been proposed for wound care, regenerative medicine-based methods have garnered the most attention. As is well known, regenerative medicine's three primary tools are gene/cell therapy, biomaterials, and tissue engineering. Multifunctional biomaterials composed of synthetic and natural components are highly advantageous for exosome carriers, which utilizing them is an exciting wound healing method. Recently, stem cell-secreted exosomes and certain biomaterials have been identified as critical components of the wound healing process, and their combination therapy appears to produce significant results. This paper presents a review of literature and perspectives on the use of stem cell-derived exosomes and biomaterials in wound healing, particularly chronic wounds, and discusses the possibility of future clinical applications. [ABSTRACT FROM AUTHOR]

Intraoperative radiotherapy (IORT) could abrogate cancer recurrences, but the underlying mechanisms are unclear. To clarify the effects of IORT-induced wound fluid on tumor progression, we treated breast cancer cell lines and human-derived tumor spheroids in 2D and microfluidic cell culture systems, respectively. The viability, migration, and invasion of the cells under treatment of IORT-induced wound fluid (WF-RT) and the cells under surgery-induced wound fluid (WF) were compared. Our findings showed that cell viability was increased in spheroids under both WF treatments, whereas viability of the cell lines depended on the type of cells and incubation times. Both WFs significantly increased sub-G1 and arrested the cells in G0/G1 phases associated with increased P16 and P21 expression levels. The expression level of Caspase 3 in both cell culture systems and for both WF-treated groups was significantly increased. Furthermore, our results revealed that although the migration was increased in both systems of WF-treated cells compared to cell culture media-treated cells, E-cadherin expression was significantly increased only in the WF-RT group. In conclusion, WF-RT could not effectively inhibit tumor progression in an ex vivo tumor-on-chip model. Moreover, our data suggest that a microfluidic system could be a suitable 3D system to mimic in vivo tumor conditions than 2D cell culture. [ABSTRACT FROM AUTHOR]

Background: The increasing need for therapeutic monoclonal antibodies (mAbs) entails the development of innovative and improved expression strategies. Chromatin insulators have been utilized for the enhancement of the heterologous proteins in mammalian cells. Methods and results: In the current study the Ccnb1ip1 gene insulator element was utilized to construct a novel vector system for the expression of an anti-CD52 mAb in Chinese hamster ovary (CHO) cells. The insulator containing (pIns-mAb) and control (pmAb) vectors were generated and stable cell pools were established using these constructs. The expression level in the cells created with pIns-mAb vector was calculated to be 233 ng/mL, and the expression rate in the control vector was 210 ng/mL, which indicated a 10.9% increase in mAb expression in pIns-mAb pool. In addition, analysis of mAb expression in clonal cells established from each pool showed a 10% increase in antibody productivity in the highest mAb producing clone derived from the pIns-mAb pool compared to the clone isolated from pmAb pool. Conclusions: More studies are needed to fully elucidate the effects of Ccnb1ip1 gene insulator on recombinant therapeutic protein expression in mammalian cells. The combination of this element with other chromatin-modifying elements might improve its augmentation effect which could pave the way for efficient and cost-effective production of therapeutic drugs. [ABSTRACT FROM AUTHOR]

Currently, nanoscale materials and scaffolds carrying antitumor agents to the tumor target site are practical approaches for cancer treatment. Immunotherapy is a modern approach to cancer treatment in which the body's immune system adjusts to deal with cancer cells. Immuno-engineering is a new branch of regenerative medicine-based therapies that uses engineering principles by using biological tools to stimulate the immune system. Therefore, this branch's final aim is to regulate distribution, release, and simultaneous placement of several immune factors at the tumor site, so then upgrade the current treatment methods and subsequently improve the immune system's handling. In this paper, recent research and prospects of nanotechnology-based cancer immunotherapy have been presented and discussed. Furthermore, different encouraging nanotechnology-based plans for targeting various innate and adaptive immune systems will also be discussed. Due to novel views in nanotechnology strategies, this field can address some biological obstacles, although studies are ongoing. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) are widely employed as invaluable therapeutics for a vast number of human disorders. Several approaches have been introduced for the improvement of mAb production in Chinese hamster ovary (CHO) cells due to the increasing demand for these products. In this regard, various chromatin-modifying elements such as insulators have been incorporated in the expression vectors to augment mAb expression. In this study, human gamma-satellite insulator containing vectors were utilized for the expression of an anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) mAb in CHO-K1 cells. To this aim, dual expression vectors encoding the antibody light chain (LC) and heavy chain (HC) with or without the insulator element were constructed, and mAb expression was evaluated in transient and stable expression. Based on the results, mAb expression significantly increased in the stable cell pool, and clonal cells developed using the human gamma-satellite insulator. In contrast, transient antibody expression was not affected by the insulator element. Finally, the enhancement of LC and HC mRNA levels was found in the insulator containing stable cell pools using the quantitative real-time-polymerase chain reaction (qRT-PCR). Our findings showed the positive effect of the human gamma-satellite insulator on the stable expression of an anti-PCSK9 immunoglobulin G1 (IgG1) mAb in CHO-K1 cells using dual expression vectors. [ABSTRACT FROM AUTHOR]