An improved cryosection method for polyethylene glycol hydrogels used in tissue engineering

The high water content of hydrogels allows these materials to closely mimic the native biological extracellular conditions, but it also makes difficult the histological preparation of hydrogel-based bioengineered tissue. Paraffin-embedding techniques require dehydration of hydrogels, resulting in substantial collapse and deformation, whereas cryosectioning is hampered by the formation of ice crystals within the hydrogel material. Here, we sought to develop a method to obtain good-quality cryosections for the microscopic evaluation of hydrogel-based tissue-engineered constructs, using polyethylene glycol (PEG) as a test hydrogel. Conventional sucrose solutions, which dehydrate cells while leaving extracellular water in place, produce a hydrogel block that is brittle and difficult to section. We therefore replaced sucrose with multiple protein-based and nonprotein-based solutions as cryoprotectants. Our analysis demonstrated that overnight incubation in bovine serum albumin (BSA), fetal bovine serum (FBS), polyvinyl alcohol (PVA), optimum cutting temperature (OCT) compound, and Fisher HistoPrep frozen tissue-embedding media work well to improve the cryosectioning of hydrogels. The protein-based solutions give background staining with routine hematoxylin and eosin, but the use of nonprotein-based solutions PVA and OCT reduces this background by 50%. These methods preserve the tissue architecture and cellular details with both in vitro PEG constructs and in constructs that have been implanted in vivo. This simple hydrogel cryosectioning technique improves the methodology for creation of good-quality histological sections from hydrogels in multiple applications.