Cell spray deposition techniques for the needs of tissue engineering are being actively developed during the last few years. They permit easier deposition of cells on surfaces with complex configurations, creation of cell multilayers and three-dimensional cell cultures. Objectives: The aim of the present study was to optimize the parameters of cell spray deposition (cell concentration, cell environment during the spraying process, and functionalization of surfaces) in order to achieve better cell adhesion, improved cell vitality and cell proliferation on the surface. Methods: Two cell lines were used in the experiments: human pulp fibroblasts isolated from fleshly extracted teeth and immortalized oral keratinocytes, TERT-2 OKF-6 (BWH Cell Culture and Microscopy Core, Boston, MA, USA). The method of non-automatic nebulization in sterile conditions was used for cell deposition. Surfaces were functionalized prior to cell spraying with either different configurations of poly-lysine (PLL)/poly-glutamic acid (PGA) multilayers or with a collagen film (Collagen Type I from calf skin). Cell characterization was performed at 24, 72 hours and 7 days after deposition using fluorescence microscopy (Nikon TE 2000), FDA/PI vital cell coloration and atomic force microscopy (AFM- MFP3D Asylum Research) in tapping and/or contact mode Results: Fluorescence microscopy images recorded on the seventh day after cell deposition proved cell survival after the spraying and evidenced the pattern of cell vitality for this period. Cell surface imaging and force mapping via AFM in force-volume mode provided the characteristics and variations of cell morphology and cell elasticity. These measurements confirmed cell adhesion on the functionalized surfaces and enabled identifying the optimal surface functionalization for an improved cell adhesion and proliferation. Conclusion: Cell spraying is an innovative method for cell deposition onto different types of surfaces that can constitute a valuable technique in cell engineering.