A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids.

The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment, but detailed cell death assays for 3D cultures (including those for ferroptosis) are scarce. In this work, we show that a new cell death analysis method, named 3D Cell Death Assay (3DELTA), can efficiently determine different cell death types including ferroptosis and quantitatively assess cell death in tumour spheroids. Our method uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. After optimization of Sytox concentration, Triton X-100 concentration and timing, we showed that the 3DELTA method was able to detect signals from all cells without the need to disaggregate spheroids. Moreover, in this work we demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. In conclusion, 3DELTA is a more cost-effective way to identify and measure cell death type in 3D cultures, including spheroids.

Pectin-bioactive glass self-gelling, injectable composites with high antibacterial activity.

The present work focuses on the development of novel injectable, self-gelling composite hydrogels based on two types of low esterified amidated pectins from citrus peels and apple pomace. Sol-gel-derived, calcium-rich bioactive glass (BG) fillers in a particle form are applied as delivery vehicles for the release of Ca2+ ions to induce internal gelation of pectins. Composites were prepared by a relatively simple mixing technique, using 20% w/v BG particles of two different sizes (2.5 and <45 µm). Smaller particles accelerated pectin gelation slightly faster than bigger ones, which appears to result from the higher rate of Ca2+ ion release. µCT showed inhomogeneous distribution of the BG particles within the hydrogels. All composite hydrogels exhibited strong antibacterial activity against methicilin-resistant Staphylococcus aureus. The mineralization process of pectin-BG composite hydrogels occurred upon incubation in simulated body fluid for 28 days. In vitro studies demonstrated cytocompatibility of composite hydrogels with MC3T3-E1 osteoblastic cells.

Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level.

BACKGROUND AND OBJECTIVE: The chronic metabolic disorder diabetes mellitus is an important cause of morbidity and mortality due to a series of common secondary metabolic complications, such as the development of severe, often slow healing skin lesions. In view of promoting the wound-healing process in diabetic patients, this preliminary in vitro study investigated the efficacy of green light emitting diode (LED) irradiation on fibroblast proliferation and viability under hyperglycemic circumstances. MATERIALS AND METHODS: To achieve hyperglycemic circumstances, embryonic chicken fibroblasts were cultured in Hanks' culture medium supplemented with 30 g/L glucose. LED irradiation was performed on 3 consecutive days with a probe emitting green light (570 nm) and a power output of 10 mW. Each treatment lasted 3 min, resulting in a radiation exposure of 0.1 J/cm2. RESULTS: A Mann-Whitney U test revealed a higher proliferation rate (p = 0.001) in all irradiated cultures in comparison with the controls. CONCLUSION: According to these results, the effectiveness of green LED irradiation on fibroblasts in hyperglycemic circumstances is established. Future in vivo investigation would be worthwhile to investigate whether there are equivalent positive results in diabetic patients.

Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation.

BACKGROUND AND OBJECTIVE: As Light Emitting Diode (LED) devices are commercially introduced as an alternative for Low Level Laser (LLL) Therapy, the ability of LED in influencing wound healing processes at cellular level was examined. STUDY DESIGN/MATERIALS AND METHODS: Cultured fibroblasts were treated in a controlled, randomized manner, during three consecutive days, either with an infrared LLL or with a LED light source emitting several wavelengths (950 nm, 660 nm and 570 nm) and respective power outputs. Treatment duration varied in relation to varying surface energy densities (radiant exposures). RESULTS: Statistical analysis revealed a higher rate of proliferation (p < 0.001) in all irradiated cultures in comparison with the controls. Green light yielded a significantly higher number of cells, than red (p < 0.001) and infrared LED light (p < 0.001) and than the cultures irradiated with the LLL (p < 0.001); the red probe provided a higher increase (p < 0.001) than the infrared LED probe and than the LLL source. CONCLUSION: LED and LLL irradiation resulted in an increased fibroblast proliferation in vitro. This study therefore postulates possible stimulatory effects on wound healing in vivo at the applied dosimetric parameters.