Measurements of elastic modulus in neuroblastoma cells under different biological and physical conditions using the atomic force microscope

Abstract

Background. Mechanobiology is a rapidly emerging field that studies the impact of physical forces on cell differentiation, physiology, and disease. Neuroblastoma cells are commonly used in neurological cell models to study Alzheimer's, Parkinson's Diseases, and Hereditary Sensory Neuropathy Type l A (HSN- l A). The latter is a neurodegenerative disease affecting the peripheral sensory neurons. This study aims to quantify the elastic modulus of neuroblastomas, used as a model for HSN- 1 A, under different biological and physical conditions. Materials and Methods. Human neuroblastoma SH-SY5Ycells (non-transfected, NT), their mutants Cl 33Y, C 133W, and wild type (WT) cells were cultured following a standard protocol. Cells were then probed by an Atomic Force Microscope (AFM) lo measure Young's modulus using the following parameters: relative selpoinl force = 0.5 nN, sample rate= l 024 Hz, tip velocity= 2 I¼m/second, extend time= 7.5 seconds and extended z-length = 15 I¼m. AFM cantilever SICON 450 I¼m with pyramidal tip was calibrated against a circular disk (diameter= 2.4cm) via the thermal noise method. Other non-transfected neuroblastomas cells were stimulated electrically with 10 I¼A for 1 hour before measuring their elastic modulus. Results. The elastic modulus of the two mutant groups (C 133Y and C 133W) were the lowest: 114 ± l O and 82 ± 11 Pa, respectively (p < 0.001, n = 120, One-way ANOVA). The nontransfected cell line had the highest elastic modulus (518 ± 58 Pa], followed by the Wild type cells (164 ± 20 Pa). Indentation analysis shows that both mutant cells had the highest indentation depth (2045 ± 178 nm and 24 14 ± l 21 nm, respectively; p < 0.00 l, n = 120, One-way ANOVA) if compared to non-transfected and Wild Type cells (963 ± 243 nm and 1553 ± 119 nm, respectively). The cells stimulated with current had the highest elastic modulus compared to non-stimulated cells (1421 ± 155 Pa and 302 ± 31 Pa, respectively; p < 0.00 l, n = 90, One-way ANOVA). An inverse relationship between the elastic modulus and indentation depth was found even in this case (1469 ± 97 nm and 703 ± 61 nm, respectively; p < 0.001, n = 90, One-way ANOVA). Conclusion. The mutations Cl 33Y and C 133W resulted in a significant decrease in membrane stiffness when compared to non-transfected cells. On the other hand, the electrical stimulation of non-transfected cells enhanced the elastic modulus concerning non-stimulated cells.

Date of Award	2019
Original language	English