3D Culture Histology Cryosectioned Well Insert Technology Preserves the Structural Relationship between Cells and Biomaterials for Time‐Lapse Analysis of 3D Cultures. Issue 11 (2nd August 2019) - Charbonneau, André M.
Abstract : When performing histology of softer biomaterials, aspiration disrupts the cellular and molecular location information. This study aims to develop a cryosectionable well insert able to preserve the biomaterial and cell's original 3D conformation from the well to histology analysis. The well insert is composed of a paraffin‐coated gelatine pill. Within the coated capsule, the human epithelial cell line (NS‐SV‐AC) is cultured in Matrigel, GrowDex, Myogel, Myogel + GrowDex, or cell culture media for 14 days. At 0 and 14 days, the samples are frozen in liquid nitrogen and cryotome is used to create sections. The slides are stained by Sirius Red and immunohistochemistry using antibodies human collagens I–V and human Ki‐67. Sirius Red shows pink shades of biomaterials and the best cellular vertical distribution throughout the sagittal section of the well is achieved with Matrigel, GrowDex, and Myogel + GrowDex; in Myogel and media, the cells sink. For collagen protein expression, only Matrigel induces a notable difference while in the other materials, collagen staining is weak or difficult to distinguish from endogenous collagens. Ki‐67 expression is maintained over time. The 3D‐cryo well insert provides a new time‐lapse histology perspective of analysis for liquid or gel cultures that maintains cells and macromolecules in their unaltered in‐well configuration. Abstract : A new cell culture plate well insert is developed for the cryohistology analysis of soft 3D cultures. The 3D‐cryo well insert maintains cell and secreted proteins in their unaltered biomaterial architecture. Histology techniques can provide information on events occurring in the developing complex environment. It is a new high‐resolution time‐lapse analysis technique for soft 3D cell cultures.