A Novel Serum-Free 2D and 3D Culture System Comprising Two Small Molecules for Culturing Mouse Lacrimal Gland Epithelial Cells

The lacrimal gland (LG) is essential for producing the aqueous layer of the tear film, which is crucial for maintaining ocular surface homeostasis¹. Dysfunction of the LG, caused by injury or inflammation, results in aqueous-deficient dry eye disease (ADDE). This condition can lead to severe ocular surface inflammation, chronic corneal disease, and, in severe cases, permanent vision loss². Current treatments for ADDE primarily manage symptoms, without addressing the underlying glandular dysfunction, which limits their long-term efficacy¹. The development of targeted therapies for LG dysfunction presents several challenges, including the lack of long-term, simple in vitro models, which limit the understanding of LG pathophysiology and hinder the development of effective therapies. While cell transplantation has shown potential in promoting LG regeneration, the limited expansion capacity of cells in vitro and the complexity of existing culture systems present significant obstacles to clinical application^1,3. Therefore, establishing a simple, long-term culture system for adult mouse LGs is essential for advancing understanding of LG physiology and pathology, as well as providing a reliable source of LG stem/progenitor cells for LG injury repair and regeneration.

Existing in vitro culture systems for LG have made significant progress, but still face notable limitations. Many of these methods rely on serum-supplemented media, which introduces several issues such as batch-to-batch variability, undefined components, and the risk of fibroblast or mesenchymal cell contamination^4,5,6,7,8. Moreover, serum-based systems are unsuitable for studying the pathogenic mechanisms of LG diseases and pose challenges for cell transplantation therapies due to potential immune rejection risks⁹. In response to these limitations, several serum-free methods have been developed for culturing LG epithelial cells (LGECs) from both mice and humans. For example, Ueda et al. successfully employed a serum-free method to culture LGECs from newborn mice¹⁰. However, this method was limited by the inability to pass the cells through subcultures and the need for a large number of neonatal glands to obtain sufficient LGECs. Similarly, Kobayashi et al. developed a serum-free culture system with cholera toxin, but faced difficulties in maintaining cellular morphology during passage¹¹. Recent advancements include Zhang et al.'s development of a 3D culture system for mouse LG stem cells and Bannier-Hélaouët et al.'s LG organoid culture system for both mice and humans^12,13,14,15. However, these systems rely on complex media formulations with multiple small molecules, growth factors, and additives, complicating the culture process and limiting scalability. These challenges highlight the need for a simplified, effective serum-free culture system that can facilitate the efficient expansion of LGECs, while maintaining their proliferative and stem/progenitor characteristics for further therapeutic applications and research.

In recent years, the rapid development of small molecule-mediated chemical reprogramming has introduced new strategies for maintaining and expanding primary adult cells in vitro¹⁶. By regulating intracellular signaling pathways, cell-matrix interactions, and cell adhesion, small molecules significantly enhance cell proliferation and plasticity, improving cell expansion efficiency and fate control¹⁷. Due to their controllable production, low immunogenicity, and non-genomic integration, small molecules are ideal for constructing in vitro systems for expanding adult epithelial cells¹⁶. Combinations of different small molecules have been shown to effectively maintain the in vitro expansion of various primary cell types, including skin, corneal, and conjunctival epithelial stem cells^18,19,20. Therefore, developing a small molecule-based strategy for expanding LGECs shows potential for future applications in both research and therapeutic settings.

A simple and efficient serum-free culture system was developed in this protocol using two small molecules, Y27632 and SB431542 (2C), to support the expansion of LGECs. By combining the advantages of these two molecules, a serum-free system was established for both 2D and 3D cultures. LGECs cultured with 2C exhibited high proliferative capacity and stem/progenitor cell characteristics, maintaining typical epithelial cell morphology after at least 10 passages in vitro. In the 3D culture, LGECs not only retained stem/progenitor cell features but also exhibited the ability to further differentiate into secretory structures after the removal of 2C, forming microglandular structures with secretory function. This serum-free system is suitable for in vitro models of LG physiology and pathology, while also providing a substantial source of cells for LG tissue engineering and regenerative applications. However, the present protocol only provides a preliminary exploration of 3D culture. Long-term 3D culture is beyond the scope of this research at this stage. It is important to note that this method is designed specifically for adult mouse LGECs and may not be applicable to other species without further optimization.

All experiments were performed in compliance with the regulations of the Association for Research in Vision and Ophthalmology (ARVO) and the Experimental Animal Ethics Committee of Jilin University and Xiamen University.

NOTE: All cell culture procedures were performed in a UV-sterilized biosafety cabinet in the cell operation room.

1. Primary culture of LGECs

1. Isolation of LG tissue
   1. Euthanize anesthetized C57BL/6J mice (6-8 weeks old) by cervical dislocation and disinfect the ear region (around the LG) with 75% ethanol to avoid contamination.
   2. Make an incision from the outer canthus to the skin beneath the ear to expose the LG. Using sterile smooth forceps and scissors, carefully separate the LG from the surrounding tissue (parotid glands and connective tissue) under a dissection microscope to obtain the LG.
   3. Place the LG in a pre-cooled tissue collection medium (DMEM with 10% Penicillin-Streptomycin-Amphotericin) in a 1.5 mL microcentrifuge tube, keeping the tissue on ice to minimize degradation.
2. Tissue preparation
   1. Transfer the LGs into a new cold tissue collection medium in a 3.5 cm dish.
   2. Remove any residual mouse fur, then peel off the connective tissue capsule surrounding the LGs. Isolate the glandular tissue by using smooth forceps to remove the connective tissue between the lobules.
      NOTE: Ensure that the capsule on the surface of the LG and the connective tissue between the lobules are thoroughly removed to expose the ducts between the lobules, which will facilitate the penetration of the digestion solution during the subsequent digestion process.
   3. Transfer the tissue into a new collection medium and wash for 5-10 min to prevent contamination.
3. Enzymatic digestion
   1. Place the LGs into a 1.5 mL microcentrifuge tube containing digestion solution (serum-free medium supplemented with 2 mg/mL Dispase II and 2 mg/mL Collagenase A). (Supplementary Table 1).
      NOTE: 1 mL of digestion solution is required for 4 LGs.
   2. Finely dissect the LG tissue using microscissors into small pieces (approximately 0.5-1 mm³) within the tube.
   3. Incubate the microcentrifuge tube at 37 °C with 5% CO₂ for 2.5-3 h. Every 30-60 min, gently mix the tissue in the digestion solution to ensure thorough enzymatic digestion.
4. LGECs isolation and harvesting
   1. Shake the microcentrifuge tube several times upon completion of the digestion time. Pipette the digestion solution several times to break the tissue into single cells or small cell clumps. Observe under a microscope to confirm the digestion progress.
      NOTE: When no visible particles remain in the digestion solution and large numbers of single cells, as well as duct-like cell aggregates, are observed under the microscope, stop the digestion process.
   2. Centrifuge the tube at 300 x g for 5 min at room temperature, then discard the digestion medium.
   3. Resuspend the cell pellet in 1 mL of sterile DPBS and centrifuge again at 1000 x g for 3 min at room temperature to remove any remaining digestive enzymes.
   4. Repeat step 1.4.3, remove the supernatant, and resuspend the cell pellet in 500 µL of serum-free medium.
5. LGECs seeding and primary culture
   1. Prepare the LGECs culture medium by supplementing serum-free medium with 1% penicillin-streptomycin and 2C (10 µM Y27632 and 10 µM SB431542). (Supplementary Table 1)
   2. Seed the cells into standard, untreated 24-well culture plates at a density of 7.5 x 10³ cells/cm² using serum-free 2C medium.
   3. After seeding, gently tap the edges of the culture plate to ensure even cell distribution. Place the plate in a 37 °C incubator with 5% CO₂.
   4. Change the culture medium for the first time 72 h after seeding. Thereafter, change the medium every 2-3 days.
      NOTE: Avoid disturbing the culture during the first 72 h. For the first medium change, do not wash the cells with DPBS. Simply remove the old medium and add fresh medium.

2. Subculturing of LGECs

NOTE: Subculture when the cell confluence reaches 80-90%, which typically occurs approximately 14 days after primary culture.

1. Remove the culture medium and gently wash the cells twice with sterile DPBS.
2. Add 250 µL of 0.25% Trypsin-EDTA and incubate at 37 °C with 5% CO₂ for 10-15 min. Once the tight junctions dissociate and the cells round up, add an equal volume of defined Trypsin Inhibitor Solution to terminate digestion.
   NOTE: If flat and large adherent cells are observed during primary culture, treat the culture with 0.25% Trypsin-EDTA for 5 min to remove these heterogeneous cells. Then, treat the culture again with 0.25% Trypsin-EDTA for an additional 5 min to further digest and obtain a purer population of LGECs for subsequent passage.
3. Mix the cell suspension by pipetting with a 1 mL pipette, then transfer to a 1.5 mL centrifuge tube and centrifuge at 1000 x g for 3 min at room temperature. Discard the supernatant.
4. Wash the cells with sterile DPBS and centrifuge again to collect the LGECs.
5. Resuspend the LGECs in 2C culture medium and seed at a 1:2 ratio for subculture. Gently mix the cells by shaking the culture plate and incubate at 37 °C with 5% CO₂.
   NOTE: Subculturing at a 1:2 ratio means that, once cell confluence reaches 80-90%, the LGECs are digested from one well plate and evenly distributed into two new well plates.
6. Change the medium on day 3 post-seeding, and then change the medium every 2-3 days, depending on cell condition.
7. Observe cell morphology daily under an inverted phase contrast microscope and document with photographs.

3. Three-dimensional (3D) culture of LGECs

NOTE: The 3D culture experiment uses P1 LGECs. When the P1 LGECs reach 80-90% confluence after 7 days of culture with 2C, they are used for 3D culture.

1. Preparation steps
   1. Transfer matrix gel (Supplementary Table 1) from the -20 °C freezer to a 4 °C refrigerator for overnight thawing.
   2. Place the required pipette tips in the -20 °C freezer to ensure proper pre-cooling.
   3. Preheat the 24-well culture plate by placing it in a 37 °C, 5% CO₂ incubator 2 h before starting the 3D culture.
   4. Prepare an ice box and place several 1.5 mL microcentrifuge tubes in it for pre-cooling. Sterilize the ice box in a biosafety cabinet before use.
2. 3D culture of LGECs
   1. Obtain P1 LGECs, as described in steps 2.1 to 2.4.
   2. Resuspend the cell pellet in 2C culture medium, and adjust the cell density to 1 × 10⁴ cells per 10 µL.
   3. Take the thawed matrix gel from the 4 °C refrigerator and the pre-cooled pipette tips from the -20 °C freezer, and place them in the ice box inside the biosafety cabinet.
   4. Remove the preheated 24-well culture plate from the incubator and place it in the biosafety cabinet.
   5. Add an appropriate volume of the cell suspension, mixed with matrix gel in a 1:1 ratio, using pre-cooled pipette tips. Mix gently to ensure uniform distribution.
   6. Pipette 20 µL of the mixture from step 3.2.5 carefully into the center of each well of the preheated 24-well plate.
   7. Invert the plate and incubate it in a 37 °C, 5% CO₂ incubator for 30 min to allow the matrix gel to solidify.
   8. Once the matrix gel has solidified, add 600 µL of 2C culture medium to each well.
   9. Incubate the culture plate in a 37 °C, 5% CO₂ incubator for 14 days. Change the medium on day 3 after seeding and every 2-3 days thereafter, depending on the cell condition.
   10. Observe 3D spheroid formation daily under an inverted phase contrast microscope and capture images for documentation.
3. Differentiation of 3D LGEC spheroids
   1. Culture 3D LGEC spheroids as described in step 3.2.
   2. After 7 days of culture, remove the 2C culture medium and gently wash the cells twice with DPBS to remove residual 2C. Take care not to disturb the matrix gel.
   3. Add serum-free medium without 2C (Null) and continue culture for an additional 7 days to induce differentiation.
   4. Change the medium every 2-3 days during the culture period.
   5. Observe the morphological changes of the induced 3D spheroids daily under an inverted phase contrast microscope and capture images for documentation.

4. Embedding and frozen sectioning of differentiated 3D LGEC spheroids

NOTE: Differentiation of 3D spheroids is induced as described in section 3.3. After 7 days of differentiation, follow the steps below for optimal cutting temperature (OCT) embedding and frozen sectioning of the differentiated 3D spheroids.

1. Aspirate the culture medium along the edges of the well. Gently wash the differentiated spheroids three times with sterile DPBS.
2. Add 1 mL of 4% paraformaldehyde (PFA) to the well in a biosafety cabinet or fume hood and fix the spheroids at room temperature for 10 min.
3. Wash the spheroids gently three times with sterile DPBS, each wash lasting 10 min, to remove the fixative.
4. Use a spatula to carefully scrape the 3D spheroid-matrix gel dome-like structure and transfer it into a 5 mL microcentrifuge tube containing 20% sucrose solution.
5. Incubate the tube overnight at 4 °C until the dome structure settles at the bottom of the tube.
6. Remove the dome structure from the sucrose solution, carefully remove any residual sucrose, and embed it in OCT compound. Freeze in liquid nitrogen and store at -80 °C.
7. Before staining, prepare 10 µm thick sections using a freezing microtome.

5. Secretion function detection of differentiated 3D LGEC spheroids

1. Sample collection
   1. Induce differentiation of 3D spheroids as described in section 3.3.
   2. After 7 days of differentiation, remove the culture medium and wash the 3D spheroids three times with DPBS.
   3. Add 500 µL of serum-free medium without 2C and incubate at 37 °C with 5% CO₂ for 2 h.
   4. Collect the culture medium and transfer it into a 1.5 mL microcentrifuge tube as a baseline sample to measure the lactoferrin (LTF) concentration before pilocarpine stimulation.
   5. Wash the 3D spheroids gently three times with sterile DPBS.
   6. Add 500 µL of serum-free medium containing 1 µg/mL pilocarpine and incubate the 3D spheroids at 37 °C with 5% CO₂ for 24 h.
   7. After incubation, collect the culture medium into a 1.5 mL microcentrifuge tube (for measuring LTF concentration after pilocarpine stimulation).
   8. Centrifuge the collected culture medium samples at 4 °C at 1000 × g for 20 min. Collect the supernatant and store it at -80 °C for later use.
2. Sample dilution
   1. Retrieve the samples from the -80 °C freezer and allow them to thaw completely at room temperature.
   2. Take 5 µL of the sample and dilute it with 495 µL of sample dilution buffer from the enzyme-linked immunosorbent assay (ELISA) kit to prepare a 100-fold dilution.
3. Reagent preparation
   1. Bring all reagents of the ELISA kit to room temperature 20 min before use.
   2. Prepare the wash buffer: Calculate the required amount based on the number of samples, and dilute the concentrated washing buffer with distilled water (1:24).
   3. Prepare the standard working solution:
      1. Centrifuge the lyophilized standard at 10,000 × g for 1 min. Add 1 mL of Reference Standard and Sample Diluent, let it stand for 10 min, and invert gently several times.
      2. After it fully dissolves, mix thoroughly with a pipette. This reconstitution produces a working solution of 2000 pg/mL.
   4. Make serial dilutions according to the kit instructions to prepare the following standard concentration gradient: 2000, 1000, 500, 250, 125, 62.5, 31.25, 0 pg/mL.
   5. Prepare the biotinylated detection antibody working solution:
      1. Calculate the required volume for the assay (50 µL/well). Centrifuge the concentrated biotinylated detection antibody at 800 × g for 1 min, then dilute the 50× concentrated biotinylated detection antibody to a 1× working solution using biotinylated detection antibody diluent.
      2. Prepare the solution just before use, making slightly more than the calculated amount, typically adding 100-200 µL more than needed.
   6. Prepare the horseradish peroxidase (HRP) conjugate working solution:
      1. Calculate the required volume for the experiment (50 µL/well). Centrifuge the concentrated HRP conjugate at 800 × g for 1 min, then dilute it to a 1x working concentration using HRP conjugate diluent.
      2. Prepare the solution just before use, making slightly more than the calculated amount, typically adding 100-200 µL more than needed.
4. ELISA assay procedure
   1. Determine the wells for the diluted standard, blank, and samples, with 3 replicates for each. Add 25 µL of the standard working solution, blank, or diluted sample solution into the corresponding wells. Cover the plate with the sealer provided in the kit and incubate at 37 °C for 90 min.
      NOTE: Solutions should be added to the bottom of the micro ELISA plate well. Avoid touching the inside wall and causing foaming as much as possible.
   2. Decant the liquid from the wells without washing. Immediately add 50 µL of the biotinylated detection antibody working solution to each well. Cover the plate with a new sealer and incubate at 37 °C for 60 min.
   3. Decant the liquid from the wells and add 350 µL of prepared wash buffer to each well. Soak for 1 min, then aspirate or decant the liquid from each well and pat it dry with clean absorbent paper. Repeat this wash step 3 times.
      NOTE: Do not allow the wells to dry out during the washing process.
   4. Add 50 µL of the prepared HRP conjugate working solution to each well. Cover the plate with a new sealer and incubate at 37 °C for 30 min.
   5. Decant the liquid from the wells and repeat the wash process five times with wash buffer as described in step 5.4.3.
   6. Add 50 µL of substrate reagent to each well. Cover the plate with a new sealer and incubate at 37 °C in the dark for 15 min.
   7. Add 25 µL of stop solution to each well to terminate the reaction. Ensure the stop solution is added in the same order as the substrate solution.
   8. Determine the optical density (OD value) of each well immediately at 450 nm using a microplate reader.

6. RNA isolation

1. Sample collection
   1. For LG tissue
      1. Isolate the extraorbital LG tissue from adult mice as described in section 1.1.2-1.1.2.
      2. Immerse the freshly isolated LG tissue in PBS to wash off any blood.
      3. Blot the surface moisture of the LG with filter paper and place the tissue into a 1.5 mL microcentrifuge tube containing 1 mL of RNA Extraction Reagent (Supplementary Table 1), keeping the tube on ice.
         NOTE: Perform grinding steps before RNA extraction to ensure complete lysis of the tissue.
      4. Add two clean steel grinding beads to the microcentrifuge tube, then place the tube in a pre-cooled high-speed low-temperature grinder.
      5. Set the grinder parameters: frequency 70 Hz, grinding time 45 s, pause time 15 s, and repeat for 10 cycles. Tighten the grinder's cap handle and start grinding.
      6. Place the tube on ice or store it at -80 °C for subsequent RNA extraction once the grinding process is complete.
   2. For LGECs
      1. Remove the culture medium and wash the cells three times with sterile DPBS when the LGECs reach 80-90% confluence.
      2. Add 600 µL of RNA extraction reagent to each well (for a 24-well plate) and incubate at room temperature for 5-10 min.
      3. Use a 1 mL pipette to repeatedly pipette the RNA extraction reagent solution in each well to ensure complete cell lysis.
      4. Transfer the solution into a 1.5 mL microcentrifuge tube.
      5. Place the tube on ice or store it at -80 °C for subsequent RNA extraction.
   3. For 3D LGEC spheroids
      1. Collect undifferentiated and differentiated 3D LGEC spheroids cultured for 14 days as described in steps 3.2-3.3. Add 1 mL of digestion solution (serum-free medium containing 1 mg/mL Dispase II) to each well, ensuring the 3D spheroid-matrix gel dome-like structure is fully covered. Incubate at 37 °C with 5% CO₂ for 30 min.
      2. Transfer the digestion solution containing the 3D spheroids into a 1.5 mL microcentrifuge tube using a 1 mL pipette. Centrifuge at 1000 × g for 3 min to collect the spheroid pellet.
      3. Wash the pellet with 1 mL of DPBS and centrifuge at 1000 × g for 3 min to collect the pellet again.
      4. Add 600 µL of RNA extraction reagent (per 20 µL of matrix gel) and incubate at room temperature for 5-10 min.
      5. Mix thoroughly using a 1 mL pipette to ensure complete lysis of the 3D spheroids.
      6. Place the tube on ice or store it at -80 °C for subsequent RNA extraction.
2. RNA extraction
   NOTE: Wear appropriate personal protective equipment (lab coat, gloves, mask) and perform all procedures involving chloroform and isopropanol in a fume hood. Dispose of chemical waste streams in designated hazardous waste containers labeled for hazardous waste generation, following institutional waste disposal protocols.
   1. Add chloroform (1/5 volume of RNA extraction reagent) to the centrifuge tube containing the RNA sample in RNA Extraction Reagent (from step 6.1).
      NOTE: Perform the procedure in a fume hood.
   2. Mix thoroughly using a vortex and incubate at room temperature for 10 min until phase separation.
   3. Centrifuge at 14,000 x g for 10 min at 4 °C.
      NOTE: After centrifugation, the liquid will separate into three layers, with RNA in the uppermost transparent aqueous phase.
   4. Prepare a new 1.5 mL RNase-free centrifuge tube and add isopropanol (1/2 the volume of RNA extraction reagent) in a fume hood.
   5. Pipette the uppermost transparent aqueous phase (1/2 the volume of RNA extraction reagent) carefully into the new RNase-free tube.
      NOTE: Be careful to avoid contaminating the middle layer when pipetting.
   6. Invert the tube several times to mix and incubate at room temperature for 10 min. Then centrifuge again at 14,000 x g for 10 min at 4 °C.
   7. Discard the supernatant and add 500 µL of 75% ethanol to wash the pellet by inverting the tube. Centrifuge again at 14,000 x g for 5 min at 4 °C.
   8. Repeat step 6.2.7. Discard the supernatant and perform a quick centrifugation to remove any residual liquid.
   9. Place the tube on ice in the fume hood, open the lid, and allow it to air dry for 10-20 min until the white pellet becomes semi-transparent.
   10. Add 20 µL of RNase-free water to dissolve the pellet. Measure RNA concentration and purity using a spectrophotometer.
       NOTE: If the RNA concentration is too high, dilute with an appropriate amount of RNase-free water.
   11. Place the RNA solution on ice or store it at -80 °C for the subsequent reverse transcription process.

7. Quantitative RT-PCR (qRT-PCR)

1. Reverse transcription
   1. Add the following components in sequence to the PCR reaction tube according to the manufacturer's instructions: 800 ng of total RNA (calculate the volume based on its concentration), 4 µL of 5x All-in-One SuperMix for q-PCR, 1 µL of gDNA remover, and RNase-free water to bring the total volume to 20 µL.
   2. Gently mix the components. Set the PCR machine to the following conditions: 55 °C for 6 min, 80 °C for 1 min, and then 12 °C indefinitely.
   3. Store the cDNA at -20 °C or keep it on ice for the qRT-PCR process after the reverse transcription reaction is complete.
2. qRT-PCR
   1. Prepare the qRT-PCR pre-mix solution for targeting different genes as follows: 5 µL of 2x Green qPCR SuperMix, 0.2 µL of Forward Primer (10 µM), 0.2 µL of Reverse Primer (10 µM), and 3.6 µL of nuclease-free water. Mix gently and place on ice.
   2. Add 9 µL of the qRT-PCR pre-mix (prepared in step 7.2.1) and 1 µL of cDNA (prepared in step 7.1) into the qRT-PCR tubes according to the experimental design.
   3. Centrifuge the tubes at 1000 × g at room temperature for 2 min.
   4. Place the tubes into the fluorescence quantitative PCR machine for detection.
      NOTE: Use a three-step method with the following program according to the reagent manufacturer's instructions: 94 °C for 30 s, 94 °C for 5 s, 60 °C for 15 s, and 72 °C for 10 s. Start cycling from step 2 for 40-45 cycles.
   5. Perform quantitative analysis using the comparative CT method (2^-ΔΔCt).
      NOTE: Each dataset should be repeated at least three independent times to ensure the reliability of the results.

8. Immunofluorescence staining

NOTE: Immunofluorescence staining is performed on LGECs cultured in 24-well plates with confluence rates of 70-80% or on frozen sections of differentiated LGECs 3D structures.

1. For LGECs
   1. Remove the culture medium from the wells and wash the cells three times with PBS.
   2. Add 500 µL of 4% PFA to each well in a biosafety cabinet or fume hood and incubate at room temperature for 15 min to fix the cells.
   3. Wash the cells three times with PBS, each wash lasting 5 min.
   4. Add 500 µL of 0.2% Triton X-100 to cover the cells and incubate at room temperature for 20 min to permeabilize the cell membrane.
   5. Wash the cells three times with PBS, each wash lasting 10 min.
   6. Remove the PBS and use an immunohistochemistry marker to draw a circle around each well of the 24-well plate. Add 50 µL of 2% BSA to each well to cover the cells. Incubate at room temperature for 1 h for blocking.
   7. Remove the BSA and add 50 µL of the diluted primary antibody (Supplementary Table 1) to cover the LGECs. Incubate at 4 °C for 16-18 h.
   8. Remove the primary antibody and repeat step 8.1.5.
   9. Add 50 µL of fluorescent secondary antibody to each well to cover the LGECs. Incubate at room temperature in the dark for 1 h.
   10. Repeat step 8.1.5, avoiding exposure to light.
   11. Add 50 µL of 4',6-diamidino-2-phenylindole (DAPI) solution to cover the LGECs and observe the staining under a fluorescence microscope.
2. For 3D LGEC spheroids
   1. Prepare the 3D LGEC spheroid frozen sections as described in step 4. Perform the immunofluorescence staining as outlined in steps 8.1.2-8.1.11.

Establish a serum-free LGECs culture system using 2C
In this protocol, the aim was to establish a simpler, more efficient culture system for expanding primary mouse LGECs. After extensive screening, a serum-free 2C combination, composed solely of Y27632 and SB431542, was successfully developed, enabling long-term in vitro expansion of LGECs. After mixed enzyme digestion, primary LGECs began to adhere and form multiple cell clones after 3 days of culture with 2C (data not shown). The first 3 days are a crucial period for LGEC culture; disturbance during this period should be avoided, as it significantly impacts clone formation and results in failed expansion of LGECs. After 12 days of culture with 2C, the clones gradually fused, and the cells displayed a well-organized epithelial morphology with tight cell-cell connections. In contrast, the control group (Null), without 2C, showed only a few adherent cells with irregular morphology (Figure 1A). Compared to the Null group, 2C significantly enhanced the proliferative capacity and stemness characteristics of LGECs, as evidenced by a higher proportion of Ki67-positive cells and increased expression of K14 (Figure 1B,C). Furthermore, qRT-PCR results showed that, compared to normal LG tissue, primary LGECs cultured with 2C exhibited higher expression of epithelial progenitor/stem cell markers (K14, P63, K15, K5) and the proliferation marker Ki67 (Figure 1D). Conversely, the expression of the differentiation marker AQP5 was nearly negligible (Figure 1D). These results indicate that the 2C serum-free culture strategy effectively maintains the stemness and proliferative capacity of LGECs.

2C supports the sustained expansion of LGECs in 2D culture
To investigate the long-term effects of 2C on maintaining the proliferative capacity of LGECs, subculturing of primary cells (P0) cultured with 2C was performed. Primary LGECs cultured with 2C maintained their morphological characteristics after 10 passages in vitro. Cells at each passage exhibited a well-organized, cobblestone-like epithelial morphology (Figure 2A). Even after more passages (P15), LGECs retained strong proliferative capacity, displayed clonal growth, and preserved epithelial morphology21. Immunofluorescence staining showed that the fluorescence intensity of the stemness marker K14 and the number of Ki67-positive proliferating cells in different passages (P3, 5, 8, 10) were comparable to those of P1 cells (Figure 2B,C). qRT-PCR results further demonstrated that the cells maintained high expression of stem/progenitor cell markers (K14, P63, K5, K15) and low expression of differentiation markers (AQP5 and α-SMA) across different passages, with minimal variation across different passages (Figure 2D). Additionally, cells from later passages (P6-P10) were cryopreserved. After 4 months of storage, they exhibited clonal growth upon thawing, maintained the typical cobblestone-like morphology, and demonstrated continued passage and expansion capability21. These results suggest that 2C supports the long-term expansion of LGECs in vitro. Even after multiple passages and cryopreservation, LGECs effectively maintain their stemness and proliferative capacity.

Establish a three-dimensional (3D) culture of LGECs using 2C
To better simulate the in vivo characteristics of the LG, LGECs were further cultured in a 3D system. Direct digestion of glandular tissue for 3D culture may result in contamination by non-epithelial cells. Therefore, in this study, relatively pure P1 LGECs were used and seeded in a low-growth-factor matrix gel for 3D culture. First, the effect of 2C on 3D spheroid formation was investigated. After 14 days of 3D culture, the number and size of the 3D spheroids were significantly increased in the 2C-treated group (+) compared to the control group without 2C (-) (Figure 3A). The mean diameter of the spheroids in the 2C-treated group was approximately 150 µm after 14 days of culture, with some variability in size. The distribution of spheroids was found to be relatively uniform. Additionally, the 2C group exhibited stronger stemness and proliferative capacity compared to the control group (Figure 3B). Thus, 2C was used for the in vitro expansion of 3D spheroids.

After 3 days of culture with 2C, cells began to aggregate into small clusters that resembled spheroid structures. After 7 days, the clusters expanded, and by day 14, they had formed more substantial structures (Figure 3C). qRT-PCR analysis showed that, compared to freshly isolated mouse LG tissue, the 3D spheroids cultured for 14 days exhibited high levels of stem/progenitor cell markers (K14, P63, K15, K5) and the proliferation marker Ki67, while the expression of the LG maturation marker AQP5 was nearly undetectable (Figure 3D). These results demonstrate that the 2C culture system not only effectively maintains the proliferative capacity and stem/progenitor cell characteristics of LGECs in 2D culture but also supports proliferation and stemness maintenance in 3D culture.

Induce differentiation of 3D cell spheroids cultured with 2C
The primary function of the LG is tear secretion, which makes the establishment of 3D LG cultures with secretory function essential. In 2D culture, various methods have been employed to induce differentiation of LGECs, such as the addition of serum, lower concentrations of EGF and bFGF, and Ca2+ supplementation. Removal of 2C has also been investigated as an induction method21. Compared to these approaches, removal of 2C most effectively induced LGECs to express the maturation marker AQP5 and the secretory function marker LTF21.

Therefore, in the 3D culture system, differentiation was induced by removing 2C. After 7 days of culture with 2C, the 3D spheroids were cultured for an additional 7 days after 2C removal. Results showed that the internal structure of individual spheroids became denser after 7 days without 2C (Figure 4A). Additionally, the differentiated spheroids aggregated and formed microglandular structures with complex ducts-like structures (red arrows in Figure 4A). qRT-PCR analysis revealed that, compared to continuous 2C culture, the induced differentiation group (Null) showed significantly reduced expression of stemness and proliferation markers (K14, P63, K15, K5, Ki67). In contrast, the expression of differentiation and secretory function markers, such as α-SMA (myoepithelial cell marker), K19 (ductal cell marker), AQP5, SOX10 (essential for forming secretory units in exocrine glands), and LTF, were significantly increased (Figure 4B). Immunofluorescence staining of the differentiated 3D spheroids revealed that AQP5 and α-SMA-positive cells were primarily located at the periphery of the spheroids, while K19-positive cells were scattered in other regions, indicating the presence of multiple LG cell types (Figure 4C). The secretory function of the differentiated microglandular structures was evaluated by pilocarpine stimulation. After 24 h, LTF levels in the culture medium were significantly elevated, confirming their secretory function (Figure 4D).

In conclusion, LGECs cultured with 2C exhibit high proliferative capacity and stemness, and are capable of forming 3D spheroids with these characteristics under in vitro conditions. Moreover, upon removal of 2C, the 3D spheroids can be induced to differentiate and self-assemble into microglandular structures with diverse cell types and secretory functions.

Figure 1: Establishment and characterization of LGECs with enhanced stemness and proliferative capacity by 2C. (A) Comparison of LGEC morphology on day 12 with and without 2C. (B,C) Immunofluorescence staining of K14 (green) and Ki67 (green), with nuclei counterstained using DAPI (blue). (D) mRNA expression levels of genes related to stemness (K14, p63, K5, K15), proliferation (Ki67), and differentiation (AQP5) were compared between primary LGECs (P0) cultured with 2C and normal LG tissues (n = 3). Statistical significance was assessed using a two-tailed Student's t-test. Data are presented as mean ± SD; **P < 0.01, ***P <0.001. This figure has been adapted with permission from Zeng et al.21. Please click here to view a larger version of this figure.

Figure 2: 2C supports continuous expansion of LGECs in vitro. (A) Cellular morphology of LGECs at different passages (P1-P10) cultured with 2C. (B) Immunofluorescence staining of K14 (green) and Ki67 (green) in LGECs from different passages (P1, 3, 5, 8, 10), with nuclei stained using DAPI (blue). (C) Statistical analysis of the fluorescence intensity of K14 and the number of Ki67-positive cells at specific passages (P1, 3, 5, 8, 10), n = 4-7. (D) Comparison of mRNA expression levels of stemness markers (K14, P63, K5, K15) and differentiation markers (AQP5, α-SMA) in LGECs from different passages (P1, 3, 5, 8, 10) cultured with 2C, compared to normal LG tissues, n = 3. * indicates a statistically significant difference between LG and LGECs of P1, 3, 5, 8, 10; # indicates a statistically significant difference between P1 and P3, 5, 8, 10. Statistical significance was assessed using a two-tailed Student's t-test. Data are presented as mean ± SD; *, #P < 0.05, **, ##P < 0.01, ***, ###P < 0.001, ****P < 0.0001, ns: no statistical significance. This figure has reprinted with permission from Zeng et al.21. Please click here to view a larger version of this figure.

Figure 3: Establishment and characterization of three-dimensional (3D) culture of LGECs with 2C. (A) Representative bright-field microscopy images of 3D cell spheroids derived from P1 cells cultured without (-) or with 2C (+) for 14 days. (B) qRT-PCR results comparing the mRNA expression levels of the proliferation and progenitor/stemness markers (Ki67, P63, K15, K5) in 3D cell spheroids cultured without (-) and with 2C (+) on day 14, n = 3. (C) Representative images of 3D cell spheroids derived from P1 single cells under 2C culture conditions at day 0, day 7, and day 14. (D) qRT-PCR analysis of mRNA expression levels of genes related to proliferation and stemness (Ki67, K14, P63, K15, K5) and differentiation markers (AQP5) in 3D cell spheroids (3D-Sph) cultured with 2C for 14 days and normal LG tissue, n = 3. Statistical significance was assessed using a two-tailed Student's t-test. Data are presented as mean ± SD; *P < 0.05, ***P < 0.001, ****P < 0.0001. This figure has been adapted with permission from Zeng et al.21. Please click here to view a larger version of this figure.

Figure 4: Differentiation of 3D cell spheroids cultured with 2C. (A) Representative bright-field microscopy images of 3D cell spheroids cultured continuously with 2C or after removal of 2C for differentiation. Red arrows indicate the duct-like structures between the 3D spheroids after differentiation. (B) Comparison of mRNA expression levels of genes related to proliferation and stemness (Ki67, K14, P63, K15, K5) and LG lineage-specific and functional markers (AQP5, SOX10, α-SMA, K19, LTF) in 3D cell spheroids with and without differentiation, n = 3. (C) Immunofluorescence staining for AQP5 (green), K19 (green), and α-SMA (green) in differentiated microglandular structures. Nuclei were counterstained with DAPI (blue). (D) ELISA analysis of LTF protein concentration in the culture medium of differentiated microglandular structures following 24 h of pilocarpine stimulation, n = 3. Statistical significance was assessed using a two-tailed Student's t-test. Data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001. This figure has been adapted with permission from Zeng et al.21. Please click here to view a larger version of this figure.

As described in the Introduction, several serum-free culture systems for LG cells have been developed in recent years^10,11,12,13. However, many of these methods have limitations, including poor cell proliferation and the inability to achieve large-scale expansion in vitro^10,11. Furthermore, recent serum-free culture systems for LG stem cells and organoids have relied on complex formulations that involve a multitude of additives, making the culture conditions unnecessarily complicated^12,13,14,15. For example, Zhang et al. used a combination of EGF, FGF10, Wnt3A, and Y27632, along with several cell culture supplements, to promote the expansion of LG stem cells in 3D culture. However, this method does not effectively achieve directed differentiation through simple adjustments in growth factors or environmental conditions^12,15. Similarly, Bannier-Hélaouët et al. expanded mouse LG organoids using a mixture of factors, including B27, Glutamax, HEPES, N-acetylcysteine, nicotinamide, Noggin, R-spondin 3, A83-01, PGE2, FSK, and FGF10. However, this method involved complex signaling pathways regulation and poses challenges in terms of achieving precise control over LG cell fate^13,14.

In contrast, the protocol presented here offers a novel, simplified serum-free culture system using only two small molecules-Y27632 and SB431542 (2C)-for the long-term expansion of LGECs. By combining the advantages of these two small molecules, this system streamlines the cell expansion process, eliminating the need for multiple additives and reducing the complexity of the culture conditions. The use of 2C allows precise control over the proliferation and differentiation of LGECs, providing an efficient and convenient approach for LG research and regeneration applications.

Several critical steps in this protocol ensure optimal culture results. First, the simplification of the digestion procedure, which previously involved multiple enzymes and filtration steps, is a key improvement^4,5,11. Only two enzymes, Dispase II and Collagenase A, are used for LG digestion, which simplifies the procedure. However, this process requires careful dissection of the LG tissue. It is especially important to remove the connective tissue, including the LG capsule and the fibrous connective tissue within and between the lobules, to ensure complete exposure of ducts within the lobules. After removing the connective tissue, the tissue should be meticulously divided into fragments to ensure complete digestion and successful isolation of LGECs. Do not directly cut the LG after extraction without removing the capsule to begin digestion, as this will prevent the digestion solution from adequately penetrating and lead to culture failure. After digestion, it is crucial to assess the tissue morphology to confirm the success of the digestion process. If the digestion solution contains mostly clumps of cells rather than individual cells with good motility, it indicates incomplete tissue processing. In this case, the issue can be addressed by more thoroughly removing the connective tissue and finely mincing it before digestion.

Another critical step is the digestion time, which is crucial for obtaining high-quality LGECs. A 2.5 to 3h digestion time, combined with manual shaking and microscope monitoring, allows for optimal cell dissociation. Digestion should be stopped when there are no visible particles remaining in the digestion solution and large numbers of single cells, as well as some duct-like cell aggregates, are observed under the microscope. After digestion, the cells should be pipetted several dozen times to ensure complete dissociation. It is important to count the cells before cell seeding. Normally, 6-8 × 10⁴ cells can be obtained from one LG of a 6-8 weeks old male mouse. A low cell count indicates incomplete digestion. In this case, the digestion time should be extended and the cells should be thoroughly dissociated by pipetting. Another factor that can contribute to culture failure is residual digestion enzymes. Before seeding the cells, wash them twice with DPBS to completely remove any remaining enzymes. Insufficient washing may result in poor cell adhesion after 72 h or a significant reduction in clone formation. Alternatively, the cells may detach and die over time. Incomplete or excessive digestion can negatively impact both the yield and quality of LGECs. For the first 72 h, it is important not to disturb the cells to ensure proper adhesion and clonal growth. After the first media change at 72 h, gentle handling is necessary, as the cells have not yet fully adhered to the plate. Do not pipette or disturb the cells during this change, as doing so may prevent them from continuing to grow and lead to culture failure. Normally, cells begin to adhere 72 h after seeding, and several small cell clones become visible under a microscope. If this does not occur as expected, the potential issues mentioned above should be addressed systematically.

While the protocol demonstrates the efficacy of 2C in expanding LGECs in 2D culture, the 3D culture system still requires further refinement. This protocol offers a preliminary exploration of 3D spheroid formation but does not yet include long-term subculture or comprehensive structural and functional assessments. Future studies should focus on characterizing the structure and receptor profiles of differentiated 3D spheroids, as well as evaluating their responses to various stimulants. Furthermore, the secretion of additional proteins, such as lysozyme, should be explored to better understand the full functional capacity of the 3D cultures. Additionally, while the effects of 2C on LGEC expansion are well-documented, the precise molecular mechanisms behind the synergistic action of Y27632 and SB431542 remain unclear. Y27632, a ROCK pathway inhibitor, has been shown to promote cell adhesion and proliferation^22,23. On the other hand, SB431542, a TGF-β pathway inhibitor, has been shown to promote cell proliferation and delay aging^24,25. However, the synergistic effects of these two molecules remain unknown. Research on the interactions of these small molecules with the ROCK and TGF-β pathways in LG physiology and pathology is important for a deeper understanding of their roles in promoting cell proliferation and differentiation. Subsequent studies will utilize sequencing technologies to investigate the specific molecular mechanisms by which 2C regulates the proliferation and differentiation of LGECs. This will improve our understanding of the LGEC culture system, facilitate more precise control of cell behavior, and identify potential targets for LG regeneration. Moreover, while the protocol has been successfully applied to mouse LGECs, its effectiveness in human LGECs has not yet been evaluated. Future studies should assess whether this serum-free culture system can be effectively applied to expand human LGECs, which would represent an important step toward clinical and translational applications.

In conclusion, this protocol presents a novel serum-free culture method, 2C, which effectively supports the expansion of LGECs in both 2D and 3D cultures while precisely regulating their proliferation and differentiation. This method overcomes the complexity and limited proliferative capacity of traditional culture systems, providing a simplified and efficient approach for research in LG physiology and pathology. Moreover, the 2C culture system offers abundant cell resources for LG regeneration and tissue engineering, presenting new potential and opportunities for the treatment of LG dysfunction and ADDE.