Effect of rice cell-derived human granulocyte-macrophage colony-stimulating factor on 5-fluorouracil-induced mucositis in hamsters.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important regulator of the maturation and function of cells in the granulocyte and macrophage lineages, and also plays a significant role in wound healing. In a previous study, we expressed human GM-CSF in rice cells (rice cell-derived human GM-CSF; rhGM-CSF). The purpose of the present study was to evaluate its effect on wound healing in oral mucositis. Oral mucositis was induced in Syrian hamster cheek pouches by 5-fluorouracil treatment and mechanical scratching. Ulcerated areas were treated from days 3 to 14 with an application of 200 µL saline, or of the same volume of a solution containing 0.04, 0.2, or 1 µg/mL rhGM-CSF. Treatment of hamsters with rhGM-CSF reduced the ulcerated areas of the oral mucosa, compared with the control. Early in the healing process, the mucositis tissue layer of the rhGM-CSF-treated group showed significantly decreased myeloperoxidase activity and increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells. Treatment with rhGM-CSF also affected expression of inflammatory cytokines in the ulcerative mucosal tissue. These results demonstrate the efficacy of plant-produced rhGM-CSF in wound healing and have significant implications for the development of rhGM-CSF as a therapeutic agent for ulcerative oral mucositis.

Enhancement of the Anticancer Ability of Natural Killer Cells through Allogeneic Mitochondrial Transfer.

An in vitro culture period of at least 2 weeks is required to produce sufficient natural killer (NK) cells for immunotherapy, which are the key effectors in hematological malignancy treatment. Mitochondrial damage and fragmentation reduce the NK cell immune surveillance capacity. Thus, we hypothesized that the transfer of healthy mitochondria to NK cells could enhance their anticancer effects. Allogeneic healthy mitochondria isolated from WRL-68 cells were transferred to NK cells. We evaluated NK cells' proliferative capacity, cell cycle, and cytotoxic capacity against various cancer cell types by analyzing specific lysis and the cytotoxic granules released. The relationship between the transferred allogenic mitochondrial residues and NK cell function was determined. After mitochondrial transfer, the NK cell proliferation rate was 1.2-fold higher than that of control cells. The mitochondria-treated NK cells secreted a 2.7-, 4.1-, and 5-fold higher amount of granzyme B, perforin, and IFN-γ, respectively, when co-cultured with K562 cells. The specific lysis of various solid cancer cells increased 1.3-1.6-fold. However, once allogeneic mitochondria were eliminated, the NK cell activity returned to the pre-mitochondrial transfer level. Mitochondria-enriched NK cells have the potential to be used as a novel solid cancer treatment agent, without the need for in vitro cytokine-induced culture.

Nanoinjection system for precise direct delivery of biomolecules into single cells.

Intracellular delivery of functional molecules such as proteins, transcription factors and DNA is effective and promising in cell biology. However, existing transfection methods are often unsuitable to deliver big molecules into cells or require carriers such as viruses and peptides specific to the target molecules. In addition, the nature of bulk processing does not generally provide accurate dose control of individual cells. The concept of single-cell-based material injection based on electrokinetic pumping through nanocapillaries could overcome these problems, yet the fabrication and operation of nanoscale 3-dimensional structures have remained unsolved. In this research, a hybrid (PDMS/glass) microfluidic chip with a true 3-dimensional nanoinjection structure (called "nanoinjection system") is presented. The nanoinjection structure was fabricated by femtosecond-laser (fs-laser) ablation in a single solid glass, which showed very successful delivery of red fluorescent protein (RFP) and expression of plasmid DNA in several different types of cells. This system is promising in that the amount of molecules to be delivered is controllable and the processed cells are systematically separated into a harvesting chamber, which can radically improve the purity of the processed cells. In addition, it was confirmed that the cells were healthy even after the molecule injection for a few seconds, indicating that the injection time can be significantly elongated, further improving the delivery efficiency of biomolecules without affecting the cell viability. We envision that the nanoinjection system having the major features of being carrier-free and dose-controllable, having an unlimited injection period, and ease of harvesting will greatly contribute to the next-generation research studies in the fields of cell biology and cell therapeutics.

Delivery of exogenous mitochondria via centrifugation enhances cellular metabolic function.

Mitochondria are essential organelles involved in the maintenance of cell growth and function, and have been investigated as therapeutic targets in various diseases. Recent studies have demonstrated that direct mitochondrial transfer can restore cellular functions of cells with inherited or acquired mitochondrial dysfunction. However, previous mitochondrial transfer methods are inefficient and time-consuming. Here, we developed a simple and easy mitochondrial transfer protocol using centrifugation, which can be applied to any cell type. By our simple centrifugation method, we found that the isolated mitochondria could be successfully transferred into target cells, including mitochondrial DNA-deleted Rho0 cells and dexamethasone-treated atrophic muscle cells. We found that mitochondrial transfer normalised ATP production, mitochondrial membrane potential, mitochondrial reactive oxygen species level, and the oxygen consumption rate of the target cells. Furthermore, delivery of intact mitochondria blocked the AMPK/FoxO3/Atrogene pathway underlying muscle atrophy in atrophic muscle cells. Taken together, this simple and rapid mitochondrial transfer method can be used to treat mitochondrial dysfunction-related diseases.

A composite dermal filler comprising cross-linked hyaluronic acid and human collagen for tissue reconstruction.

In this study, we developed a composite filler comprising cross-linked hyaluronic acid (HA) and human collagen (COL) derived from the human umbilical cord with the aim of improving its biocompatibility and longevity compared with commercially available fillers. After HA/ COL composite fillers were made in two different ratios (10:1 and 5:1), the physical properties of the fillers were evaluated. The interior morphologies and in vivo weight change of these hydrogels were also characterized at 1-16 weeks after injection into mice. To evaluate their biocompatibility and durability in vivo, we injected the composite fillers into nude mice subcutaneously. The variations of injected gel weight were measured and compared with the commercial dermal fillers (Restylane and TheraFill). The composites showed improved or similar physical properties (complex viscosity of 19-22 × 10(5) cP, and injection force of 10- 12 N) over the commercial dermal fillers. Sixteen weeks following the injection, the ratio of remaining composite filler weight to initial weight (75.5 ± 16.9%; 10:1) was shown to be greater than that of the commercial fillers (43.2 ± 8.1%, Restylane; 12.3 ± 5.3%, TheraFill). In addition, immunohistochemical analysis with angiogenesis-related markers such as isolectin and vWF revealed newly formed blood vessels and cellular influx into the composite filler, which were not observed in the other fillers. These results clearly suggest that the HA/COL composite filler is a superior candidate for soft tissue reconstruction. The filler we developed may be a suitable candidate as an injectable dermal filler for tissue augmentation in humans.