Background

The extracellular matrix (ECM) is composed of more than 300 core structural components (Burgstaller et al., 2017), whose physical and chemical features regulate crucial cellular mechanisms (Gattazzo et al., 2014) including differentiation, migration, and proliferation. Thus, the study of the ECM is essential for understanding some pathological conditions and diseases including cancer and fibrosis (Elowsson Rendin et al., 2019, Wishart et al., 2020, Júnior et al., 2021). Also, decellularized extracellular matrix (dECM) scaffolds have many potential applications in tissue engineering and regenerative medicine. In fact, dECM has been used for the generation of ECM hydrogels (Marhuenda et al., 2022) and the recellularization of whole previously decellularized organs (Ohata and Ott, 2020), as well as several applications in the regeneration of tissues (Zhu et al., 2019). Therefore, it is not surprising the increased interest in physiomimetic tissue scaffolds by producing decellularized tissue samples (Mendibil et al., 2020).

The elimination of cells from tissue to obtain the ECM is possible by using physical, chemical, enzymatic, or a combination of these approaches (Mendibil et al., 2020). Physical strategies include freeze/thawing cycles, which induce ice crystals in the matrix, disrupting the cell membrane. Chemical strategies include detergents that solubilize the cell membrane and hypertonic or hypotonic solutions causing cell disruption by osmotic shock. Finally, enzymatic approaches can target the cell’s nuclear material, such as with deoxyribonuclease (DNase), or the cell-ECM adhesion, such as with trypsin. However, the available decellularization protocols have important limitations. Many of them can take up to several days (Wishart et al., 2020, Wüthrich et al., 2020) and are still not particularly flexible or accessible since they are designed for the decellularization of full organs or thick sections (tissue blocks). This type of protocol would not suit the decellularization of many tissue samples, as is the case for clinical biopsies. In fact, clinical biopsies are scarce and cannot be decellularized by accessing the tissue’s vasculature. Since no current protocols have explored the decellularization of glass-attached tissue sections, a method that allows for the study of the exact location before and after decellularization is needed to fill this gap.

The method presented here is significantly faster and less wasteful (i.e., can produce a single acellular tissue slice, instead of requiring a large sample portion) than other available methods, while maintaining the sample’s mechanical properties and being suited for cell culture applications (Narciso et al., 2022). Additionally, it provides the option for studying the same tissue section before and after decellularization, which is invaluable for studies of certain pathologies and of scarce or valuable clinical samples. Patient biopsies, for example, are tested for several markers and histopathological features; hence, the entire sample cannot be decellularized. This method allows for studies of both native tissue and dECM to be carried out in the same sample. For early-stage tumors especially, this is of paramount importance as cancer cells are removed during decellularization and their location cannot be pinpointed. Furthermore, the tissue's architecture is preserved throughout decellularization by pre-attaching the samples to a glass slide. For tissues like bladder and lung, where organ inflation is required to emulate different conditions, this inflation can be performed on the native tissue, guaranteeing a more physiological result.