Single-cell-based measurement of supraphysiological thermal injury in carcinoma cells on a microchip

Hyperthermia affects certain regulatory proteins, kinases or cyclins, resulting in alternations to the cell cycle and even to apoptosis. Damage to the cell plasma membrane is a key factor in the killing of a cell by hyperthermia. Analysis at the single-cell level is necessary for understanding the fundamental mechanisms of hyperthermia-induced cell death and the generation of thermotolerance in surviving cells. The main purpose of this study is to fabricate a hydrogel chip with microwells for cellular patterning and to demonstrate the measurement of supraphysiological thermal injury in human carcinoma cells (HeLa cells) at the single-cell level. To accomplish this, measurement of membrane injury by dye leakage post-thermal insult was performed and reported in this work. For cell concentrations at 0.5 × 10⁶ cells/mL, the occupancy of cells on the microchip with 40 μm microwells was up to 86.6%, a value far higher than that found on the 30 μm microwells (approximately 78.5%). The fluorescent images showed that calcein leakage occurred when cell membranes were damaged under supraphysiological temperatures between 43 and 50°C. The single-cell-based experiment of thermal injury in the microchip with hydrogel microwells was therefore successfully demonstrated.