Growth of hollow cell spheroids in microbead templated chambers.

Cells form hollow, spheroidal structures during the development of many tissues, including the ocular lens, inner ear, and many glands. Therefore, techniques for in vitro formation of hollow spheroids are valued for studying developmental and disease processes. Current in vitro methods require cells to self-organize into hollow morphologies; we explored an alternative strategy based on cell growth in predefined, spherical scaffolds. Our method uses sacrificial, gelatin microbeads to simultaneously template spherical chambers within a hydrogel and deliver cells into the chambers. We use mouse lens epithelial cells to demonstrate that cells can populate the internal surfaces of the chambers within a week to create numerous hollow spheroids. The platform supports manipulation of matrix mechanics, curvature, and biochemical composition to mimic in vivo microenvironments. It also provides a starting point for engineering organoids of tissues that develop from hollow spheroids.

Knock-in of Cx46 partially rescues fiber defects in lenses lacking Cx50.

PURPOSE: Connexins 46 (Cx46) and 50 (Cx50) support lens development and homeostasis. Knockout (KO) of Cx50, but not Cx46, causes defects in lens fiber organization, F-actin enrichment, gap junction (GJ) size, ball-and-socket (BS) maturation, and GJ-associated protein distributions. To further determine the unique roles of Cx50 and Cx46, we investigated whether these defects persisted in Cx46 knock-in (Ki) lenses. Ki mice had Cx46 knocked-in to their Cx50 loci, where it was expressed under endogenous Cx50 promoters. METHODS: Fiber cell morphology and the distribution of lens membrane/cytoskeleton proteins from wild-type (WT), Ki, and Cx50 KO mice were visualized by immunofluorescent labeling and confocal microscopy. RESULTS: Cx46 Ki partially rescued Cx50 KO lens fiber defects. Three-week-old Ki lens fibers had typical F-actin distributions but were nonuniformly sized and disorganized. The Cx-associated proteins zonula occludens-1 (ZO-1) and ß-dystroglycan (ßDys) no longer localized to the nuclei but remained absent from GJs. BS formed, but this occurred with lower than WT frequency. BS appeared with greater frequency in 8-week-old Ki lenses, but so did aberrant balloon-like structures similar to those in Cx50 KO lenses. Unexpectedly, 8-week-old Cx50 KO and Ki cortical lens fibers were no longer disorganized. CONCLUSIONS: Cx identity is important for some aspects of fiber development (organization, Cx association with ZO-1 and ßDys) but not others (F-actin enrichment). Either Cx supports BS maturation, but the sparsity of BS and presence of balloon-like structures in Ki lenses suggest that Cx50 is more capable of doing so. The partial rescue of BS structures may support the rapid growth of cortical fibers to the improved growth of Ki lenses compared to Cx50 KO lenses at young ages. Neither absence of Cx50 nor presence of Ki Cx46 affects cortical fiber cell organization by the age of 8 weeks.