Magnetic bioassembly platforms for establishing craniofacial exocrine gland organoids as aging in vitro models

Abstract

A multitude of aging-related factors and systemic conditions can cause lacrimal gland (LG) or salivary gland (SG) hypofunction leading to degenerative dry eye disease (DED) or dry mouth syndrome, respectively. Currently, there are no effective regenerative therapies that can fully reverse such gland hypofunction due to the lack of reproducible in vitro aging models or organoids required to develop novel treatments for multi-omic profiling. Previously, our research group successful developed three-dimensional (3D) bioassembly nanotechnologies towards the generation of functional exocrine gland organoids via magnetic 3D bioprinting platforms (M3DB). To meet the needs of our aging Asian societies, a next step was taken to design consistent M3DB protocols to engineer LG and SG organoid models with aging molecular and pathological features. Herein, a feasible step-by-step protocol was provided for producing both LG and SG organoids using M3DB platforms. Such protocol provided reproducible outcomes with final organoid products resembling LG or SG native parenchymal epithelial tissues. Both acinar and ductal epithelial compartments were prominent (21 ± 4.32% versus 42 ± 6.72%, respectively), and could be clearly identified in these organoids. Meanwhile, these can be further developed into aging signature models by inducing cellular senescence via chemical mutagenesis. The generation of senescence-like organoids will be our ultimate milestone aiming towards high throughput applications for drug screening and discovery, and for gene therapy investigations to reverse aging.

Fig 1. Lab protocols for lacrimal gland (LG) and salivary gland (SG) organoid biofabrication via M3DB and induction of cellular senescence.

Created with BioRender.com.

Fig 2. Morphological heterogeneity of primary LG cells in monolayer cultures.

Primary cells isolated from porcine LG are cultured in expansion media for 7 days. The populations of large polygonal-like epithelial cells (A), small polygonal-like epithelial cells (B), epithelial spherule (C), dendritic cells (D), and fibroblast-like cells (E) are observed under phase-contrast microscopy at 20X of magnification. Scale bar: 200 μm.

Fig 3. Morphological and proteomic profiling of LG cells in 2D systems.

Acinar, ductal and myoepithelial/ductal progenitor compartments of the LG in 2D culture while in expansion media or EM (A). Differentiated cells after 7 days (C) showing numbers of epithelial-like cells (arrow), spindle shape cells (arrowhead) and dendritic cells (asterisk). Scale bar: 200 μm. AQP5, KRT14, and KRT19 protein markers were profiled and compared in both media conditions (EM versus EEM). Protein markers are quantified and displayed as a heat map with values as average % ±SE.

Fig 4. Micrograph morphologies of cells or organoids from LG 2D culture system to organoid stages.

Differentiated LG cells in 2D were magnetized with magnetic nanoparticle solution (MNP) and then 3D assembled by M3DB (A). Organoids were cultured and their morphology was profiled via light microscopy and high throughput scanning analysis for 8 days. Scale bar: 200 μm.

Fig 5. Epithelial compartments and representation of functional datasets in LG organoids.

Expression of acinar, ductal and progenitor epithelial markers (A). Epithelial function evaluated by Ca²⁺ uptake assays after parasympathetic stimulation with carbachol (B) and trans-epithelial electrical resistance (TEER) (C) in LG organoids. Scale bar: 200 μm.

Fig 6. Multi-omics profiling of cellular senescence-induced LG organoids.

Senescence markers are evaluated including β-galactosidase activity (A), gene expression arrays (B), and levels of RIND-EDSBs (C) after inducing the organoid with etoposide. For inducing cellular senescence in the organoid, the concentration of etoposide required to create a 50% reduction in metabolism was 10 μM as determined by ATP-luciferase activity with CellTiter-Glo® 3D kit from Promega, USA (D). Abbreviations: CTL LGO–control LG organoid with normal epithelial function; Eto-induced LGO–etoposide-induced LG organoid with impaired epithelial function.