Five-week-old female mice (C57BL/6) were used. All animal experiments were carried out following the guidelines of the Institutional Animal Care and Use Committee of Seoul National University (approval number: SNU-210105-6-4).

Isolated submandibular glands were minced with a razor blade, and the homogenate was incubated in Hanks’ Balanced Salt Solution with CaCl₂, MgSO₄ (Welgene, Gyeongsan, Korea), together with 1 mg/mL hyaluronidase (Sigma, St. Louis, MO, USA) and 0.2% collagenase II (Thermo Fisher, Waltham, MA, USA). Mechanically and enzymatically dissociated cells were incubated in 60 mm dishes at 37°C for 1 hour. Cells were neutralized with Dulbecco’s modified Eagle’s medium (DMEM; Welgene) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, USA), and centrifuged at 500×g for 3 minutes. After incubation, debris was filtered through 100 and 40 µm strainers (SPL Life Sciences Co., Ltd., Seoul, Korea) for single-cell isolation. The isolated cells were suspended in a basal medium composed of DMEM/F12 1:1 (v/v; Thermo Fisher), 20 ng/mL fibroblast growth factor-2 (Peprotech, Rocky Hill, NJ, USA), 20 ng/mL epithelial growth factor (Peprotech), 1% N-2 supplement (Thermo Fisher), 1% insulin (Cell Application, San Diego, CA, USA), 1 µM dexamethasone (Sigma), and 1% penicillin-streptomycin (Sigma). Cells were then cultured onto poly-HEMA-coated 60 mm culture dishes in a CO₂ incubator (5% CO₂ in humidified air) for 5 days.

The cells were harvested and prepared in a single-cell suspension. The total suspension (40 μL) was then mixed with 80 μL of growth factor reduced (GFR) Matrigel (Corning, NY, USA) to form the high concentration of Matrigel group, or with 40 μL of DMEM/F12 1:1 and 40 μL of GFR Matrigel to form low concentration of Matrigel group. The cells were seeded in Matrigel-pre-coated 48-well plates (SPL Life Sciences) to prepare SGC organoids. After solidifying the gels at 37°C for 20 minutes, basal medium was added, and SGC organoids were maintained for 4 days in a CO₂ incubator (5% CO₂ in humidified air). The basal medium was changed into differentiation medium composed of DMEM/F12 1:1 (v/v; Thermo Fisher), 1 mg/mL bovine serum albumin (Bovogen Biologicals Pty Ltd., Keilor East, Australia), 1% Insulin-Transferrin-Selenium (Thermo Fisher), 1% N-2 supplement (Thermo Fisher), 200 ng/mL fibroblast growth factor-1 (Peprotech), 20 ng/mL fibroblast growth factor-7 (Peprotech), 1 μM Y-27632 (Cayman Chemical, Ann Arbor, MI, USA), 1% penicillin-streptomycin (Sigma) for 8 days. The medium (200 µL/well) was added and changed every 2-3 days and maintained in a CO₂ incubator (5% CO₂ in humidified air). A total of 1 mg/mL of dispase (Life Technologies, Camarillo, CA, USA) was added and incubated at 37°C for 30 minutes to harvest the organoids. The percentage of spheroids with budding structures was measured by counting the number of spheroids with budding structures among the total number of spheroids.

Total RNA was extracted using a PureLinkTM RNA Mini Kit (Invitrogen, Camarillo, CA, USA). cDNA synthesis was performed M-MLV reverse transcriptase following the manufacturer’s protocol (M.biotech, Hanam, Korea). Real-time polymerase chain reaction (PCR) was performed using SYBR Pre-mix Ex Taq II (Takara, Tokyo, Japan) and a StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). Cycling was performed for 30 seconds at 95°C, followed by 40 amplification cycles of 5 seconds at 95°C and 30 seconds at 60°C. Gapdh was used for normalization. The primer sequences for murine genes are listed in Table 1.

All data were presented as the mean±standard deviation, and the Student t-test, as well as one-way analysis of variance followed by Tukey’s post-hoc test, was performed to determine the significance of experimental differences. Statistical analysis were performed using Prism 7.0 (GraphPad, Boston, USA). All experiments were independently repeated at least 3 times. A p-value <0.05 was considered statistically significant.

Salivary gland organoids derived from submandibular glands were generated using the paradigm illustrated in Fig. 1. Suspensions of cells were induced to form compact aggregates and then embedded in Matrigel to mimic the in vivo environment of the salivary gland. The organoids were maintained for 12 days under specific conditions, using basal medium for 4 days and differentiation medium for 8 days. The organoids were harvested on day 12 after embedding, and a series of analyses was performed.

First, we observed SGC organoids embedded in low-concentration Matrigel to assess morphological and molecular changes associated with the differentiation of embedded structures. Budding structures appeared from day 5 after organoid culture (data not shown), and interestingly, Y-27632 induced significant cell growth and reduced apoptosis compared to the control group on day 10 and day 12 (i.e., after 6 and 8 days of differentiation medium treatment) (Fig. 2A). Moreover, Y-27632 significantly increased the number of spheroids with budding structures compared to the control group (13.43±1.683 and 73.37±6.736, respectively) (Fig. 2B). The mRNA expression level of the functional marker, Amy1, was significantly upregulated in spheroids of the treated cells. However, the expression levels of the acinar marker, Mist 1, and ductal marker, Krt 8 showed a small but significant decrease. Another acinar marker, Aqp 5, and ductal progenitor marker, Ascl3, showed no significant difference between the 2 groups of cells (Fig. 2C). These results suggest that Y-27632 induces differentiation into SGC organoids in a low Matrigel concentration-based 3D culture system in terms of morphology, but whether this leads to clear differentiation into the acinar or ductal lineage is not known exactly.

Next, we observed SGC organoids embedded in high-concentration Matrigel to assess morphological and molecular changes associated with the differentiation of embedded structures compared to the low Matrigel concentration condition. As in the low-concentration Matrigel, budding structures appeared from day 5 after organoid culture (data not shown). Also, Y-27632 induced significant cell growth compared to the control group on day 10 and day 12 (i.e., after 6 and 8 days of differentiation medium treatment). Interestingly, the size of organoids in the Y-27632 treatment group was significantly smaller compared to those embedded in low-concentration Matrigel, and the budding structure was better displayed in the bright field (Fig. 3A). As noted above, Y-27632 significantly increased the number of spheroids with budding structures compared to the control group (21.5±2.784 and 75.57±2.385, respectively) (Fig. 3B). Similar to the morphology, mRNA expression levels showed a difference from the low Matrigel concentration condition. The mRNA expression of Amy1, Mist 1 and Ascl3 showed significant increases compared to the control group. However, as in the low Matrigel concentration condition, Aqp5 did not show a significant difference between the 2 groups of cells, and the expression level of Krt8 decreased slightly (Fig. 3C). These results suggest that Y-27632 clearly induces differentiation of SGC organoids in a high-concentration Matrigel compared to a low-concentration condition.