Cellular model based on laser microsurgery of cell spheroids to study the repair process

In this study, modern techniques of laser microsurgery of cell spheroids have been used to develop a new simple, reproducible model for studying the mechanisms of repair and regeneration in vitro. Nanosecond laser pulses were applied to perform a microdissection of the outer and the inner zones of the spheroids from dermal fibroblasts. To achieve effective dissection and preservation of spheroid viability, the optimal parameters were chosen: 355 nm wavelength, 100 Hz frequency, 2 ns pulse duration, laser pulses in the range of 7–9 μJ. After microdissection, we observed injury of the spheroids: the edges of the wound surface opened and the angular opening reached a value of more than 180°. As early as during the first hour after spheroid microdissection with laser radiation, the surviving cells changed their shape: cells on the spheroid surface and directly in the damaged area became rounded. One day after microdissection, the structure of the spheroids began to partially recover, the cells in the surface layers began to take the original flattened shape; debris of dead damaged cells and their fragments was gradually cleared from the spheroid composition. In the proposed model, the first data on stimulation of structure recovery of injured spheroids from dermal fibroblasts with a P199 synthetic polypeptide, which is used in cosmetology for the initiation of antiaging and regenerative effects in the skin, were received. After microdissection, recovery of the spheroids structure with a few surface layers of flattened imbricated arranged cells and polygonal cells of the inner zone in the presence of P199 peptide was faster than in the control group, and was completed within 7 days, presumably due to the remodeling of the survived cells.