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3D Culture Histology Cryosectioned Well Insert Technology Preserves the Structural Relationship between Cells and Biomaterials for Time‐Lapse Analysis of 3D Cultures
Author(s) -
Charbonneau André M.,
AlSamadi Ahmed,
Salo Tuula,
Tran Simon D.
Publication year - 2019
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201900105
Subject(s) - sirius red , histology , staining , matrigel , microbiology and biotechnology , capsule , glycosaminoglycan , biomaterial , cell culture , spheroid , chemistry , biology , biomedical engineering , cell , anatomy , in vitro , pathology , medicine , biochemistry , botany , genetics
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.