IADR Abstract Archives
Basement Membrane Assembly Establishes Polarity and Connectivity in Salivary Cells
Objectives: Our aim is to develop an autologous 3D model for reimplantation of functional salivary glands to relieve xerostomia. Primary human salivary acinar-derived cells (hSACs) reorganize into multicellular secretory units in tunable hyaluronic acid (HA)-based hydrogels. We hypothesize that cell polarity is a dynamic process associated with the synthesis and integrity of the de novo basement membrane (BM) surrounding the structure. The composition of the surrounding BM (type IV collagen, laminin, and perlecan/HSPG2) and its effect on polarity (Par3, lumen formation) were assessed during structure growth up to 15 days in culture.
Methods: BM organization and composition were demonstrated by immunofluorescence targeting type IV collagen, laminin, and perlecan/HSPG2. Par3 expression and lumen formation were assessed to determine degree of polarity in structures. Confocal microscopy and dye transfer assays (Lucifer yellow, LY) were used to determine connexin32 expression and function in 2D and 3D cultures (n=10/gp).
Results: BMs surrounding multicellular hSAC structures were found to contain type IV collagen, laminin and perlecan/HSPG2 by day 3. Cells in 2D expressed, but did not organize, these proteins. BM expression in 3D clusters of hSACs was associated with increased connectivity as assessed by LY dye transfer. Organizing structures with discontinuous BMs or irregular distributions of BM proteins did not exhibit well-defined lumens nor localized polarity associated proteins (Par3) at cell junctions.
Conclusions: When cultured in 3D HA hydrogels, hSACs reorganize into acini-like structures exhibiting similar BM proteins present in the native extracellular matrix of the BM of salivary gland tissue. BM composition and integrity, when assembled in 3D but not 2D, drives salivary acini organization, lumen formation, and polarity that influences direct as well as indirect pathways of cell-cell signaling. We conclude that culture in 3D can provide the proper cues to reestablish BM-stimulated polarity and vectorial secretion of salivary components.
Methods: BM organization and composition were demonstrated by immunofluorescence targeting type IV collagen, laminin, and perlecan/HSPG2. Par3 expression and lumen formation were assessed to determine degree of polarity in structures. Confocal microscopy and dye transfer assays (Lucifer yellow, LY) were used to determine connexin32 expression and function in 2D and 3D cultures (n=10/gp).
Results: BMs surrounding multicellular hSAC structures were found to contain type IV collagen, laminin and perlecan/HSPG2 by day 3. Cells in 2D expressed, but did not organize, these proteins. BM expression in 3D clusters of hSACs was associated with increased connectivity as assessed by LY dye transfer. Organizing structures with discontinuous BMs or irregular distributions of BM proteins did not exhibit well-defined lumens nor localized polarity associated proteins (Par3) at cell junctions.
Conclusions: When cultured in 3D HA hydrogels, hSACs reorganize into acini-like structures exhibiting similar BM proteins present in the native extracellular matrix of the BM of salivary gland tissue. BM composition and integrity, when assembled in 3D but not 2D, drives salivary acini organization, lumen formation, and polarity that influences direct as well as indirect pathways of cell-cell signaling. We conclude that culture in 3D can provide the proper cues to reestablish BM-stimulated polarity and vectorial secretion of salivary components.