Titanium Culture Vessel Presenting Temperature Gradation for the Thermotolerance Estimation of Cells.

Hyperthermia can be induced to exploit the thermal intolerance of cancer cells, which is worse than that of normal cells, as a potential noninvasive cancer treatment. To develop an effective hyperthermia treatment, thermal cytotoxicity of cells should be comprehensively investigated. However, to conduct such investigations, the culture temperature must be accurately regulated. We previously reported a culture system in which the culture temperature could be accurately regulated by employing metallic culture vessels. However, appropriate temperature conditions for hyperthermia depend on the cell species. Consequently, several experiments need to be conducted, which is a bottleneck of inducing hyperthermia. Hence, we developed a cell culture system with temperature gradation on a metallic culture surface. Michigan Cancer Foundation-7 cells and normal human dermal fibroblasts were used as cancer and normal cell models, respectively. Normal cells showed stronger thermal tolerance; this was because the novel system immediately exhibited a temperature gradation. Thus, the developed culture system can be used to investigate the optimum thermal conditions for effective hyperthermia treatment. Furthermore, as the reactions of cultured cells can be effectively assessed with the present results, further research involving the thermal stimulation of cells is possible.

Development of accurate temperature regulation culture system with metallic culture vessel demonstrates different thermal cytotoxicity in cancer and normal cells.

Hyperthermia has been studied as a noninvasive cancer treatment. Cancer cells show stronger thermal cytotoxicity than normal cells, which is exploited in hyperthermia. However, the absence of methods evaluating the thermal cytotoxicity in cells prevents the development of hyperthermia. To investigate the thermal cytotoxicity, culture temperature should be regulated. We, thus, developed a culture system regulating culture temperature immediately and accurately by employing metallic culture vessels. Michigan Cancer Foundation-7 cells and normal human dermal fibroblasts were used for models of cancer and normal cells. The findings showed cancer cells showed stronger thermal cytotoxicity than normal cells, which is quantitatively different from previous reports. This difference might be due to regulated culture temperature. The thermal stimulus condition (43 °C/30 min) was, further, focused for assays. The mRNA expression involving apoptosis changed dramatically in cancer cells, indicating the strong apoptotic trend. In contrast, the mRNA expression of heat shock protein (HSP) of normal cells upon the thermal stimulus was stronger than cancer cells. Furthermore, exclusively in normal cells, HSP localization to nucleus was confirmed. These movement of HSP confer thermotolerance to cells, which is consistent with the different thermal cytotoxicity between cancer and normal cells. In summary, our developed system can be used to develop hyperthermia treatment.

Effect of hydroxy groups and microtopography generated by a nanosecond-pulsed laser on pure Ti surfaces.

In this paper, we study a process for modifying the surface microtopography of the Ti oxide layer using a nanosecond-pulsed laser (NPL). Even now, the mechanism by which hydroxyl groups are generated on the titanium surface treated by NPL is not clear. Hence, we evaluated the surface properties of the NPL defocus distances on pure titanium surfaces, and investigated the relationship between the generation of hydroxyapatites/cell viability and the titanium surface characteristics. The NPL defocus distance was varied from 0 to 4 mm. Defocus distances of 0 and 2 mm generated microtopographical features on the titanium surface, and the resulting surfaces exhibited a greater density of OH groups than the surface treated with a defocus distance of 4 mm. The surfaces treated using defocus distances of 0 and 2 mm were found to be coated with microspherical hydroxyapatite composed of coexisting plate- and needle-like crystals after immersion in simulated body fluid, and alkaline phosphatase activity assays indicated improved cell compatibility. The improvements in biocompatibility and cell compatibility were due to the pocket-like microtopographical structures formed along the processing trace. These pockets contained a large amount of OH groups, and promoted the growth of hydroxyapatite.

Enzyme-free cell detachment mediated by resonance vibration with temperature modulation.

Cell detachment is an essential process in adherent cell culture. However, trypsinization, which is the most popular detachment technique used in culture, damages cellular membranes. Reducing cellular membrane damage during detachment should improve the quality of cell culture. In this article, we propose an enzyme-free cell detachment method based on resonance vibration with temperature modulation. We developed a culture device that can excite a resonance vibration and control temperature. We then evaluated the cell detachment ratio and the growth response, observed the morphology, and analyzed the cellular protein of the collected cells-mouse myoblast cell line (C2C12). With the temperature of 10°C and the maximum vibration amplitude of 2 µm, 77.9% of cells in number were successfully detached compared with traditional trypsinization. The 72-h proliferation ratio of the reseeded cells was similar to that with trypsinization, whereas the proliferation ratio of proposed method was 12.6% greater than that of trypsinization after freezing and thawing. Moreover, the cells can be collected relatively intact and both intracellular and cell surface proteins in the proposed method were less damaged than in trypsinization. These results show that this method has definite advantages over trypsinization, which indicates that it could be applied to subcultures of cells that are more susceptible to trypsin damage for mass culture of sustainable clinical use. Biotechnol. Bioeng. 2017;114: 2279-2288. © 2017 Wiley Periodicals, Inc.

Efficient Subculture Process for Adherent Cells by Selective Collection Using Cultivation Substrate Vibration.

Cell detachment and reseeding are typical operations in cell culturing, often using trypsin exposure and pipetting, even though this process is known to damage the cells. Reducing the number of detachment and reseeding steps might consequently improve the overall quality of the culture, but to date this has not been an option. This study proposes the use of resonant vibration in the cell cultivation substrate to selectively release adherent calf chondrocyte cells: Some were released from the substrate and collected while others were left upon the substrate to grow to confluence as a subculture-without requiring reseeding. An out-of-plane vibration mode with a single nodal circle was used in the custom culture substrate. At a maximum vibration amplitude of 0.6 µm, 84.9% of the cells adhering to the substrate were released after 3 min exposure, leaving a sufficient number of cells for passage and long-term cell culture, with the greatest cell concentration along the nodal circle where the vibration was relatively quiescent. The 72-h proliferation of the unreleased cells was 20% greater in number than cells handled using the traditional method of trypsin-EDTA (0.050%) release, pipette collection, and reseeding. Due to the vibration, it was possible to reduce the trypsin-EDTA used for selective release to only 0.025%, and in doing so the cell number after 72 h of proliferation was 42% greater in number than the traditional technique.

Inhibitory function of NKT cells during early induction phase of nickel allergy.

Until now, metal allergies have been regarded as a Th1-type immune response.　However, because the contribution of a Th2-type immune response has been suggested by clinical findings, we previously examined the Th2-type immune response during the development of metal allergies using a GATA-3 transgenic (GATA-3 Tg) mouse model. As a result, a Th2-type immunization reaction was suggested to be involved in the early phase of metal allergies. Recently, the involvement of NKT cells in metal allergies has been suggested. We examined this possibility using the activation of NKT cells and an NKT cell-deficient mouse model to determine the contribution of NKT cells to nickel allergy in the present study. In NKT cell-deficient mice, ear swelling was remarkably increased, compared with that in control mice. Also, in mice that had been treated with α-galactosylceramide (α-GalCer) to activate NKT cells, the ear swelling response was remarkably inhibited, compared with that in untreated mice. These facts show that NKT cells are involved in the inhibition of nickel allergy-induced ear swelling responses.

Control of cellular activity of fibroblasts on size-tuned fibrous hydroxyapatite nanocrystals.

We controlled the performance of L929 mouse fibroblasts using various hydroxyapatite (HA) nanocrystals, such as nanofibers, nanoneedles, and nanosheets, to better understand the effects of size and shape of the HA nanocrystals on the cells. The cellular activity on nanofibers with a diameter of 50-100 nm was significantly enhanced relative to that on a flat HA surface because large amounts of the proteins needed for adhesion and proliferation could be stored in the substrate. On the other hand, initial adhesion and subsequent proliferation were inhibited on surfaces consisting of fine nanoneedles and nanosheets with a diameter/thickness of less than 30 nm due to the limited area available for the formation of focal adhesions. These facts indicate that fibroblast activity is highly sensitive to the surface topography. Therefore, size tuning of the nanoscale units composing the substrate is essential to enhance cellular performance.

Th2 immune response plays a critical role in the development of nickel-induced allergic contact dermatitis.

BACKGROUND: The precise roles of T helper (Th)1-type and Th2-type cytokine responses in nickel (Ni)-induced allergic contact dermatitis have not yet been clearly defined. We investigated the involvement of Th2 cytokines in Ni-induced contact hypersensitivity reaction using GATA-3 transgenic (Tg) mice. METHODS: A Ni-titanium (Ti) alloy was implanted under the skin of GATA-3 Tg mice. A Ni solution was then injected 1 month after sensitization. The ear swelling response was measured at several time points after the injection; the cytokine levels in the skin were measured at 48 h after injection, and the serum levels of IgE were measured 1 month after injection. In addition, purified CD4+ splenic cells obtained from the GATA-3 Tg mice sensitized with the Ni-Ti alloy were infused into Rag-2(-/-) mice, and the ear swelling response of these mice after a further challenge with Ni solution was also measured. RESULTS: Marked ear swelling and elevated serum IgE levels and skin tissue levels of IL-4 were observed in Ni-Ti-sensitized GATA-3 Tg mice. The Rag-2(-/-) mice transfused with the CD4+ splenic cells from the Ni-Ti alloy sensitized GATA-3 Tg mice showed a significantly more pronounced ear swelling response than the control mice. CONCLUSION: We confirmed the participation of Th2-type immune reactions in Ni-induced allergy using GATA-3 Tg mice.

Adhesion of osteoblast-like cells on nanostructured hydroxyapatite.

The response of osteoblast-like cells seeded on hydroxyapatite (HAp) substrates consisting of nanosized crystals was investigated. Various types of HAp nanocrystals, such as nanofibers, nanoneedles and nanosheets, were selectively prepared as substrate through the hydrolysis of a solid precursor crystal of CaHPO(4) in alkaline solutions by varying the pH and ion concentrations. Although all the substrates were macroscopically flat and smooth, the nanoscale topography influenced cell activity, including the adhesion, proliferation, elongation and formation of actin stress fibers. The presence of fine nanoneedles and nanofibers on the surface restricted the cellular activities, while the cells steadily proliferated on a nanoscopically smooth surface of large grains and on a substrate consisting of wide nanosheets. These results suggest that the adhesion and subsequent responses of osteoblast-like cells were affected by the contact domain size between the cell and the substrate. Isolated small domains of the nanostructured HAp limited focal adhesion formation in the cells associated with the formation of stress fibers. Stable adhesion with contact domains larger than 100nm in width was suggested to be required for cell survival. On the other hand, insufficient adhesion on the fine nanoneedles was found to lead to apoptosis.