Normal LG tissues were obtained from non-damaged regions of the patients with eye-related disease. The tissues were chopped and washed with advanced DMEM/F12 (Gibco, Carlsbad, CA, USA) supplemented with 1% penicillin-streptomycin (Welgene, Gyeongsan-si, Korea) and then enzymatically digested with advanced DMEM/F12 supplemented with 0.125 mg/mL dispase II (Wako, Richmond, VA, USA), 0.1 mg/mL DNase I (MilliporeSigma, Burlington, MA, USA), 0.125 mg/ml collagenase II (Gibco, Carlsbad, CA, USA), and 1% penicillin-streptomycin for 1 h at 37 °C with shaking (150 rpm). After digestion, the supernatant was passed through a 70-μm cell strainer (SPL, Pocheon-si, Gyeonggi-do, Korea) and pelleted by centrifugation at 200g for 5 min. The pellet was resuspended in culture media and mixed with Matrigel (Corning, Corning, NY, USA) at a ratio of 1:1 (v:v), plated on a 48-well plate at a density of 1X104 per well, and incubated with 5% CO2 at 37 °C for 10 min to polymerize the matrices. The LG organoids were cultured in five different media modified from human prostate and salivary organoid culture medium [26, 27]. The components of each medium are listed in Supplementary Table 1. The culture medium was changed every two to three days.

To confirm the origin of cells forming the organoid, epithelial cell adhesion molecule (EpCAM)-positive epithelial lineage cells were sorted using the MACS method (Miltenyibiotec, USA, 130-042-201). Briefly, single cells from the lacrimal tissue were incubated with anti-EpCAM (Santacruz, CA, USA, sc-59906) for 1 h at 4 °C, washed with MACS buffer, and then incubated with anti-mouse IgG Microbeads (Miltenyibiotec, 130-048-402) for 30 min at 4 °C. After washing with PBS, the EpCAM-negative cells were passed through an MACS column, while the EpCAM-positive cells in the MACS column were isolated, washed, and then cultured in Matrigel.

Tissues and organoids were washed with D-PBS (Welgene, Korea), fixed with 4% paraformaldehyde (Bio-solution, Seoul, Korea) for 30 min, and embedded in paraffin. Paraffin sections (6-μm-thick) were deparaffinized in xylene and hydrated in a graded ethanol series. The samples were then stained with H&E, Alcian blue, PAS staining kit (Abcam, Cambridge, MA, USA), and Masson’s trichrome staining kit (Dako, Santa Clara, CA, USA) according to the manufacturers’ protocol. For immunofluorescence analysis, fixed samples were cryoprotected by immersion in PBS supplemented with 30% sucrose and 0.1% sodium azide at 4 °C. The cryoprotected samples were embedded in optimal cutting temperature (OCT, Sakura, Japan) compound, rapidly frozen in liquid nitrogen, and stored at − 80 °C until use. Sections (4-μm-thick) of the frozen block were pre-blocked with 5% normal horse serum (Vector, IL, USA) in Tris-buffered saline (Welgene) for 2 h at room temperature (RT) and incubated with primary antibody at 4 °C overnight. After washing with PBS, the sections were incubated with secondary antibody for 2 h at RT. For nuclear staining, the sections were treated with 1 ug/ml Hoechst 33342 (MilliporeSigma, Burlington, MA, USA, 1 μg/ml) for 20 min. Primary antibodies used for immunostaining included antibodies against aquaporin 5 (AQP5; Abcam), alpha-smooth muscle actin (α-SMA; Biolegend, San Diego, CA, USA), vimentin (VIM; Cell signaling, Danvers, MA, USA), lysozyme (LYZ; Diagnostic biosystems, Pleasanton, CA, USA), E-cadherin (E-CAD; Santa Cruz Biotechnology, Dallas, TX, USA), anti-BrdU (Novus, Centennial, CO, USA), and Ki67 (Abcam, Cambridge, MA, USA). The secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) used included Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 594 goat anti-mouse IgG, and Alexa Fluor 594 goat anti-rat IgG.

Total RNA was extracted from isolated tissues or organoids using MagListo™ 5 M Cell Total RNA Extraction Kit (Bioneer, Daejeon Metropolitan City, Korea) following the manufacturer’s protocol. Thereafter, 1 μg of RNA was used to synthesize cDNA using PrimeScript™ RT Master Mix (TaKaRa, Kyoto City, Japan). Quantitative RT-PCR was performed with a Thermal Cycler Dice® Real-Time System III (TaKaRa, Kyoto City, Japan) using SYBR® Premix Ex Taq™ II (TaKaRa). The PCR primers sequences are listed in Supplementary Table 2. PCR experiments were carried out in triplicates.

Ca2+ mobilization to the cytoplasm was detected using a Fluo-4 Calcium Imaging Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Briefly, the organoids were treated with Fluo-4 AM for 15 min at 37 °C, followed by incubation at RT for 15 min. After washing with PBS, the organoids were stimulated with 1 μg/mL pilocarpine (MilliporeSigma). Calcium signaling was then observed using a Nikon Eclipse Ti2 microscope (Nikon, Tokyo, Japan).

To demonstrate the secretory function of LG organoids, the lysosomal enzyme N-acetyl-β-glucosaminidase (NAG), also known as α-galactosidase B, in organoid cultured medium was detected using an NAG assay kit (MilliporeSigma) following the manufacturer’s protocol. Briefly, organoids were incubated in serum-free DMEM/F12 for 2 h, treated with pilocarpine (1 μg/mL), and then incubated at 37 °C in a 5% CO2 incubator for 24 h. The medium was collected at 2 h and 24 h after pilocarpine treatment and analyzed for NAG catalytic activity. The reaction product was detected colorimetrically at 405 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific).

The secretory proteins from organoids were detected using TEM . Briefly, the cultured organoids were washed with D-PBS and fixed with 2% glutaraldehyde-paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 12 h. After washing with 0.1 M PB, samples were post-fixed with 1% OsO4 dissolved in 0.1 M PB for 2 h, dehydrated in an ascending gradual ethanol series (50%–100%), infiltrated with propylene oxide, and embedded with a Poly/Bed 812 kit (Polysciences, PA, USA). After pure fresh resin embedding and polymerization at 65 °C in an electron microscope oven (DOSAKA, Japan) for 24 h, the Poly/Bed embedded samples were cut into approximately 70-nm-thick sections with a Leica EM UC-7 (Leica Microsystems, Wetzlar, Germany) equipped with a diamond knife (Diatome, PA, USA), transferred to copper and nickel grids, contrast-stained with 6% uranyl acetate and lead citrate (Fisher), and observed using a transmission electron microscope (JEOL, Japan) at 80 kV acceleration voltage.

To identify the proteins secreted by LG organoids after pilocarpine treatment, the culture medium was harvested 2 h after of pilocarpine treatment and analyzed through proteomics analysis [28]. In brief, proteins (200 μg) in the medium were digested using the filter-aided sample preparation (FASP) method with centrifugal filters (Millipore, MA, USA). After desalting the samples with a Sep-Pak® Vac 1 cc C18 cartridge (Waters, MA, USA), the peptides were collected, purified, and quantified via LC-MS/MS analysis. LC-MS/MS assay was performed using a Dionex Ultimate 3000 HPLC coupled with a Q Exactive™ Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Raw MS/MS data were quantified using MaxQuant (Max Planck Institute) and classified by gene ontology (GO) analysis. T test P  2,