Simple chitin-based cell culture platform for production of biopharmaceuticals.

OBJECTIVES: Gene therapy using viral vectors and antibody-based therapies continue to expand within the pharmaceutical market. We evaluated whether Cellhesion® VP, a chitin-based material, can be used as 3D culture platform for cell lines used for the production of antibodies and viral vectors. RESULTS: The results of Cell Counting Kit-8 assay and LDH assay revealed that Cellhesion® VP had no adverse effect to Human Embryonic Kidney (HEK) 293, A549 and Chinese hamster ovary (CHO) DG44-Interferon-ß (IFN) cells. Cell growth analyses showed that Cellhesion® VP supported the 3D culture of HEK293, A549 and CHO DG44- IFN-ß cells with a spherical morphology. Importantly, subculture of these cell lines on Cellhesion® VP was easily performed without trypsinization because cells readily transferred to newly added scaffold. Our data also suggest that CHO DG44-IFNß, cultured on Cellhesion® VP secreted IFNß stably and continuously during the culture period. CONCLUSIONS: Cellhesion® VP provides a simple and streamlined expansion culture system for the production of biopharmaceuticals.

A Novel 3D Culture System Using a Chitin-Based Polysaccharide Material Produces High-Quality Allogeneic Human UCMSCs with Dispersed Sphere Morphology.

Mesenchymal stem cell (MSC) transplantation, in particular allogeneic transplantation, is a promising therapy for a variety of diseases. However, before performing allograft treatment it is necessary to find suitable donors, establish culture methods that maintain cell quality, and reduce cell production costs. Here, we present a new method of producing allogeneic MSCs combining human umbilical cord-derived mesenchymal stem cells (UCMSCs) and chitin-based polysaccharide fibers (Cellhesion® MS). UCMSC numbers significantly increased, and cells grew as dispersed spheres on Cellhesion® MS. Subsequent biological analyses showed that the expression levels of stemness-related and migration-related genes were significantly upregulated, including octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), and C-X-C chemokine receptor type 4 (CXCR4). The secretion levels of paracrine factors such as prostaglandin E2 (PGE2), TNFα-stimulating gene (TSG)-6, fibroblast growth factor 2 (bFGF), and Angiogenin (Ang) from UCMSCs using Cellhesion® MS were significantly higher than with microcarrier and U-bottom plate culture. In addition, culture supernatant from UCMSCs with Cellhesion® MS had better angiogenic potential than that from monolayer cultured UCMSCs. Furthermore, we succeeded in a scaled-up culture of UCMSCs with Cellhesion® MS using a closed culture bag. Therefore, Cellhesion® MS is a key material for producing high-quality UCMSCs in a three-dimensional (3D) culture system.

STAP-2 protein promotes prostate cancer growth by enhancing epidermal growth factor receptor stabilization.

Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signaling pathways and promotes tumorigenesis in melanoma and breast cancer cells. However, the contribution of STAP-2 to the behavior of other types of cancer cells is unclear. Here, we show that STAP-2 promotes tumorigenesis of prostate cancer cells through up-regulation of EGF receptor (EGFR) signaling. Tumor growth of a prostate cancer cell line, DU145, was strongly decreased by STAP-2 knockdown. EGF-induced gene expression and phosphorylation of AKT, ERK, and STAT3 were significantly decreased in STAP-2-knockdown DU145 cells. Mechanistically, we found that STAP-2 interacted with EGFR and enhanced its stability by inhibiting c-CBL-mediated EGFR ubiquitination. Our results indicate that STAP-2 promotes prostate cancer progression via facilitating EGFR activation.

CCR7 is involved in BCR-ABL/STAP-2-mediated cell growth in hematopoietic Ba/F3 cells.

Chronic myeloid leukemia is a clonal disease characterized by the presence of the Philadelphia chromosome and its oncogenic product, BCR-ABL, which activates multiple pathways involved in cell survival, growth promotion, and disease progression. We previously reported that in murine hematopoietic Ba/F3 cells, signal transducing adaptor protein-2 (STAP-2) binds to BCR-ABL and up-regulates BCR-ABL phosphorylation, leading to enhanced activation of its downstream signaling molecules. The binding of STAP-2 to BCR-ABL also influenced the expression levels of chemokine receptors, such as CXCR4 and CCR7. For the induction of CCR7 expression, signals mediated by the MAPK/ERK pathway were critical in Ba/F3 cells expressing BCR-ABL and STAP-2. In addition, STAP-2 cooperated with BCR-ABL to induce the production of CCR7 ligands, CCL19 and CCL21. Our results demonstrate a contribution of CCR7 to STAP-2-dependent enhancement of BCR-ABL-mediated cell growth in Ba/F3 cells.

PML suppresses IL-6-induced STAT3 activation by interfering with STAT3 and HDAC3 interaction.

The promyelocytic leukemia protein PML acts as a tumor suppressor by forming transcription-regulatory complexes with a variety of repressor proteins. In the present study, we found that endogenous PML suppresses interleukin (IL)-6-induced gene expression as well as phosphorylation and transcriptional activation of STAT3 in hepatoma cells. We also found that PML-mediated suppression of IL-6-induced STAT3 activation by disrupting interactions between STAT3 and HDAC3. These results indicate that PML modulates IL-6-induced STAT3 activation and hepatoma cell growth by interacting with HDAC3.