Problems of cell visualization in three-dimensional hydrogel matrices and ways to solve them

Abstract

Synthetic hydrogels are extensively utilized as scaffolds in tissue engineering and regenerative medicine. A crucial step in evaluating their functionality involves the microscopic visualization of surface-cultured cells to assess biocompatibility, viability, and cellular functions. However, the complex physicochemical properties of hydrogels introduce significant methodological challenges for obtaining high-quality microscopic images.
During the characterization of cells cultured on the surface of the developed hydrogel, we identified several key obstacles to effective visualization: optical opacity and heterogeneity of the matrix across different z-planes, which hinders the ability to focus on cells; material autofluorescence, leading to a reduced signal-to-noise ratio in fluorescence microscopy; non-specific binding of gel components to fluorescent dyes and antibodies; and limited diffusion of antibodies and other large molecules through the gel pores, which compromises the effectiveness of standard immunocytochemistry (ICC) protocols. To overcome these limitations, a series of strategies were tested, including: blocking non-specific binding sites with 5% Bovine Serum Albumin (BSA); substituting DAPI with Hoechst stain for nuclear staining; and employing rigorous negative controls to validate ICC protocols. The use of cells stably expressing fluorescent proteins is proposed as a promising alternative strategy to circumvent artifacts related to reagent penetration and non-specific binding.
This work summarizes the identified visualization challenges and provides an analysis of the tested solutions, offering a framework to improve the reliability and quality of cellular imaging within complex three-dimensional hydrogel matrices.

Speaker

Salina Ksenia
Centre for Soft Matter and Physics of Fluids, Bauman Moscow State Technical University, Moscow, Russia
Russia

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