Improved cell culture techniques from 2D to 3D have provided models that more closely mimic the native physiological condition (gene expression, cellular functions and cell behavior1). The TrueGel3D™ hydrogel system has a complete set of reagents to design chemically defined biomimetic hydrogel that can recapitulate natural extracellular matrix (ECM) environments and provides valuable insight into cellular differentiation and morphogenesis.
Madin Darby Canine Kidney (MDCK) cells are epithelial cells derived from kidney tissue of an adult female cocker spaniel. These cells serve as models to study epithelial morphogenesis. The objective of this study is to evaluate TrueGel3DTM hydrogel in growing MDCK cells and form spherical cysts.
Some cells need attachment sites for growing in a chemically defined environment. The RGD peptide, composed of arginine, glycine and aspartic acid have been identified as a mediator of cell-substratum and cell-cell interactions. Integrins on the cell surface recognize RGD motif and support cell adhesion. In this MDCK cell cyst formation study, we also investigate the optimum concentration of RGD peptide to study epithelial morphogenesis. The scrambled RGD peptide, that does not know to mediate cell adhesion, is used as a control.
TrueGel3D™ hydrogel preparation
All steps are performed in sterile hood and the volume ratio of each component is added as indicated below.
Chemical cell fixation and confocal microscopy
Figure 1.Cyst counted from confocal images from day 15 samples. Cyst formation increases with increasing concentration of TrueGel3DTM RGD integrin adhesion peptide.
Figure 2.Merged, confocal images of cyst formed with MDCK cells after 15 days of culture in TrueGel3D™ FAST-PVA hydrogel modified with (A) 500 µmol/L of TrueGel3D™ RGD integrin adhesion peptide, (B) 500 µmol/L of scrambled RGD peptide and (C) 500 µmol/L of thioglycerol. Green: nuclei; Red: actin cytoskeleton. Scale bar: 200 µm; Magnification: 60X
TrueGel3D™ hydrogel FAST-PVA with PEG nondegradable crosslinker can grow and form cysts for MDCK cell lines and generate in-vitro models to study epithelial morphogenesis and polarity. Cyst morphology of single layer polarized cells enclosing a lumen is comparable to that observed in natural collagen. TrueGel3D™ RGD integrin adhesion peptide specifically mediate cell adherence to promote cyst formation.
Cysts with polarized cells share many characteristics including physiological development of epithelial tissues and confocal microscopy. These shared similarities have proved to be a powerful tool to analyze 3D structures of MDCK colonies. Similar hydrogel systems can be used to study pulmonary alveolar function by growing alveolar-like cysts (ALCs)2 and to investigate the etiological role of extracellular matrix proteins in polycystic kidney diseases3.