Cell isolation and organoid formation from human lacrimal gland tissue
Lacrimal gland tissues were obtained from patients and harvested from non-damaged regions. Tissues were chopped and washed with advanced DMEM/F12 (Gibco, Carlsbad, CA, USA) containing 1% penicillin-streptomycin (Welgene, Gyeongsan-si, Korea) and then enzymatically digested with advanced DMEM/F12 containing 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 hour 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 spinning down. The pellet was resuspended in culture media and mixed with Matrigel (Corning, Corning, NY, USA) at a ratio of 1:1 (v:v), plated onto a 48-well plate at a density of 1X104 per well, and incubated with 5% CO2 at 37°C for 10 min for polymerization of matrices. Lacrimal gland organoids were cultured in five different media modified from human prostate and salivary organoid culture medium [26,27]. Components of each medium are listed in Supplementary Table 1. The culture medium was changed every 2-3 days.
To confirm the origin of cells forming the organoid, EpCAM-positive cells known as epithelial stem cells were sorted using the MACS method (Miltenyibiotec, 130-042-201). Briefly, single cells from lacrimal tissue were incubated with anti-EpCAM (Santacruz, sc-59906) for 1 hour at 4°C, washed with MACS buffer, and then incubated anti-mouse IgG Microbeads (Miltenyibiotec, 130-048-402) for 30 min at 4°C. After washing with PBS, EpCAM negative cells were passed through a MACS column. EpCAM positive cells in the MACS column were isolated, washed, and then cultured in Matrigel.
Histology and immunofluorescence
Tissues and organoids were washed with D-PBS (Welgene, Gyeongsan-si, Korea), fixed with 4% paraformaldehyde (Bio-solution, Seoul, Korea ) for 30 min, and embedded in paraffin. Paraffin sections of 6 μm in thickness were deparaffinized in xylene and hydrated in a graded series of ethanol. These 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 their respective manufacturer’s protocol. For immunofluorescence analysis, fixed samples were cryoprotected by immersing in PBS containing 30% sucrose and 0.1% sodium azide at 4C. The cryoprotected samples were embedded in optimal cutting temperature (OCT, Sakura) compound, rapidly frozen in liquid nitrogen, and stored at -80C until use. Sections (4 μm in thickness) of the frozen block were pre-blocked with 5% normal horse serum (Vector) in Tris-buffered saline (Welgene, Gyeongsan-si, Korea) for 2 hours at room temperature (RT) and incubated with primary antibody at 4°C overnight. After washing with PBS, sections were incubated with secondary antibody for 2 hours at RT. For nuclear staining, Hoechst 33342 (MilliporeSigma, Burlington, MA, USA, 1 ug/ml) treatment was performed for 20 min. Primary antibodies used for immunostaining included Aquaporin5 (Abcam, Cambridge, MA, USA), α-SMA (Biolegend, San Diego, CA, USA ), Vimentin (Cell signaling, Danvers, MA, USA), Lysozyme (Diagnostic biosystems, Pleasanton, CA, USA), E-cadherin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-BrdU (Novus, Centennial, CO, usa), and Ki67 (Abcam, Cambridge, MA, USA). 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 isolation and quantitative RT-PCR
Total RNAs were from isolated tissues or organoids using MagListo™ 5M Cell Total RNA Extraction Kit (Bioneer, Daejeon Metropolitan City, Korea ) following the manufacturer’s protocol. Then 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, Kyoto City, Japan). Sequences of PCR primers are listed in Supplementary Table 2. PCR experiments were carried out in triplicate.
Calcium flux assay with Fluo-4
Mobilization of Ca2+ to the cytoplasm was detected using a Fluo-4 Calcium Imaging Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. Briefly, organoids were treated with Fluo-4 AM for 15 min at 37C followed by incubation at RT for 15 min. After washing with PBS, organoids were stimulated with pilocarpine of 1 μg/ml (MilliporeSigma, Burlington, MA, USA). Calcium signaling was then observed using a Nikon Eclipse Ti2 microscope (Nikon, Tokyo, Japan).
To demonstrate the secretory function of lacrimal gland organoids, lysosomal enzyme N-acetyl-β-glucosaminidase (NAG), also known as α‐galactosidase B, in organoid cultured medium was detected using a NAG assay kit (MilliporeSigma, Burlington, MA, USA) following the manufacturer’s protocol. Briefly, organoids were incubated in serum-free DMEM/F12 for 2 hours, treated with pilocarpine (1 ug/ml), and then incubated at 37C in a 5% CO2 incubator for 24 hours. The medium was collected at 2 hours and 24 hours after pilocarpine treatment and analyzed for NAG catalytic activity with a NAG assay kit. Reaction product was detected colorimetrically at 405 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific, Waltham, MA, USA).
Transmission electron microscope analysis
To detect secretory proteins from organoids, transmission electron microscope (TEM) analysis was performed. Briefly, cultured organoids were washed with D-PBS and fixed with 2% glutaraldehyde-paraformaldehyde in 0.1M phosphate buffer (PB, pH7.4) for 12 hr. After washing with 0.1M PB, samples were post-fixed with 1% OsO4 dissolved in 0.1 M PB for 2 h, dehydrated in an ascending gradual series (50-100%) of ethanol, infiltrated with propylene oxide, and embedded with a Poly/Bed 812 kit (Polysciences). After pure fresh resin embedding and polymerization at 65°C in an electron microscope oven (DOSAKA) for 24 h, Poly/Bed embedded samples were cut into about 70 nm thick sections and stained with 6% uranyl acetate and lead citrate (Fisher) for contrast staining. These sections were cut with a Leica EM UC-7 (Leica Microsystems) equipped with a diamond knife (Diatome) and transferred onto copper and nickel grids. All thin sections were observed with a TEM (JEOL) at an acceleration voltage of 80kV.
Proteomic analysis for secretome of lacrimal gland organoids
To identify secreted proteins by pilocarpine in lacrimal gland organoids, the culture medium was harvested after 2 hours of pilocarpine treatment and analyzed by proteomics . In brief, proteins (200 ug) in the medium were digested using filter-aided sample preparation (FASP) method with centrifugal filters (Millipore). After desalting samples with a Sep-Pak® Vac 1cc C18 cartridge (Waters), peptides were collected, purified, and quantified for 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 GO (Gene Ontology) analysis. T-test P-value of <0.05 and Fold-change (>2, <-2) were applied to determine differential expression protein (DEP) between control and pilocarpine treated groups.
Mouse dry eye model and organoid transplantation
Eight-week-old male C57BL/6 mice (Koatech, Pyeongtaek, Korea) or C57BL/6- Tg(CAG-EGFP)131Osb/LeySopJ (CAG-EGFP) mice (Nihon SLC) were used as dry eye disease model or for manipulation of lacrimal gland organoids, respectively. The experimental protocol for animal use was reviewed and approved by CHA University Institutional Animal Care and Use Committee. Lacrimal gland tissue was obtained from eGFP-Tg mouse. Organoids were formed and cultured following the same method used for human organoids. To create an inflammation-induced dye eye model, 15 μl of ConA (Concanavalin A, 10 mg/ml in PBS, MilliporeSigma, Burlington, MA, USA) was injected into the extra-orbital gland of wild type mouse lacrimal tissue. The same volume of PBS was injected for the control group. At 7 days after ConA injection, cell clumps from GFP expressed organoids (1 X 104 cells/15 μl in advanced DMEM/F12/Matrigel) were injected into the extra-orbital gland space (Fig. 5). After 2 weeks, mouse lacrimal gland tissues were harvested for immunofluorescence analysis.
Statistically significant differences were analyzed by Student’s t-test of one-way analysis of variance (ANOVA) with post hoc test Tukey for multiple comparisons using the GraphPad Prism software package, version 3.0 (GraphPad Prism). All experiments were conducted at least three times. The number of independent experiments is indicated by n. Significance was considered at p < 0.05.