3D Culture Histology Cryosectioned Well Insert Technology Preserves the Structural Relationship between Cells and Biomaterials for Time‐Lapse Analysis of 3D Cultures

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.

3D Cultures of Salivary Gland Cells in Native or Gelled Egg Yolk Plasma, Combined with Egg White and 3D-Printing of Gelled Egg Yolk Plasma

“…Previously, we described in detail the design and fabrication of the 3D-Cryo well insert [36]. Briefly, gelatine pills (size 000, Capsuline ®, Pompano Beach, FL, USA) were coated twice with paraffin (or Histowax) (00403, HistoLab, Espoo, Finland) heated to liquid, cooled, and exposed 20 min to UV lights.…”

“…Two cell-less controls were frozen and cut. See our previous publication for more details on this technique [36].…”

“…Here, we used several experiments to examine human cells’ behaviour in egg biomaterial combinations and test other egg biomaterial applications for tissue engineering. More specifically, we first hypothesized that our well insert technology [36] would provide more information on cell distribution and proliferative state. Both human SG cell types (acinar [37,38] and stromal) were cultured in similar EYP + Media and EYP + EW in the 3D-Cryo well insert and stained with Sirius Red [39] and Ki-67 [40] at 0 and 14 days.…”

Fabrication techniques of biomimetic scaffolds in three‐dimensional cell culture: A review

“…Recently, a gelatin-based 3D cryo-sectioned well-insert technology has been developed to preserve the architecture of cells and soft biomaterials (scaffolds; Charbonneau, Al-Samadi, Salo, & Tran, 2019) suitable for histological analysis. This signifies that cryo-modification may be used as a value-added technique in 3D culture to enhance the mechanical stiffness of an in-well conformation.…”

“…Advantages are numerous, however, one of the limitations during the analysis of 3D culture with scaffolds is the displacement of original conformation or disruption of information on molecular location due to external hindering mechanical forces. Recently, a gelatin‐based 3D cryo‐sectioned well‐insert technology has been developed to preserve the architecture of cells and soft biomaterials (scaffolds; Charbonneau, Al‐Samadi, Salo, & Tran, 2019) suitable for histological analysis. This signifies that cryo‐modification may be used as a value‐added technique in 3D culture to enhance the mechanical stiffness of an in‐well conformation.…”