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. Author manuscript; available in PMC: 2026 Jan 1.
Published in final edited form as: Methods Mol Biol. 2025;2848:59–71. doi: 10.1007/978-1-0716-4087-6_4

Trabecular Meshwork Regeneration for Glaucoma Treatment Using Stem Cell-Derived Trophic Factors

Ajay Kumar 1, Enzhi Yang 2, Yiqin Du 2
PMCID: PMC11971979  NIHMSID: NIHMS2058157  PMID: 39240516

Abstract

Glaucoma is one of the leading causes of irreversible blindness. Stem cell therapy has shown promise in the treatment of primary open-angle glaucoma in animal models. Stem cell-free therapy using stem cell-derived trophic factors might be in demand in patients with high-risk conditions or religious restrictions. In this chapter, we describe methods for trabecular meshwork stem cell (TMSC) cultivation, secretome harvesting, and protein isolation, as well as assays to ensure the health of TMSC post-secretome harvesting and for secretome periocular injection into mice for therapeutic purposes.

Keywords: Glaucoma, Trabecular meshwork, Stem cells, Secretome, Periocular injection

1. Introduction

Glaucoma is called the black hole of irreversible blindness and affects around 76 million people [1, 2]. Elevated intraocular pressure (IOP) due to increased resistance in the conventional outflow pathway, mainly the trabecular meshwork (TM) and Schlemm’s canal endothelium, is the main risk factor associated with primary open-angle glaucoma. In glaucoma patients, TM cellularity is reduced, leading to TM microstructure change and dysfunction resulting in excessive accumulation of the aqueous humor in the eye and increased IOP, which leads to apoptosis of retinal gangion cells (RGC) and optic nerve damage, causing irreversible blindness. Currently, the only effective target for glaucoma treatment is reducing IOP, which is achieved via topical application of eye drops and medicines like prostaglandin analogs, beta blockers, carbonic anhydrase inhibitors, Rho kinase inhibitors, and alpha agonists, as well as surgical and laser procedures. These treatments have different side effects or limitations and they don’t address the abnormal patho-physiology of the TM; hence, it is difficult to maintain long-term effects. Excitingly, new stem cell-based therapies are being developed with the promise of long-term efficacy against glaucoma.

Stem cell-based therapies based on TM regeneration have been explored and reported over the past decade as a novel treatment for glaucoma. Different stem cell types, such as trabecular meshwork stem cells (TMSCs) [3–7], adipose-derived stem cells (ADSCs) [8, 9], mesenchymal stem cells (MSCs) [10, 11], and induced pluripotent stem cells (iPSCs) [12], have been applied towards TM regeneration to treat glaucoma in vitro and in animal models. Although the results in animal models are very promising, developing stem cell-free therapies using stem cell-derived trophic factors has also gained much traction in recent years. Indeed, there is a demand for cell-free therapies for use in patients with religious restrictions or with high risk conditions that prevent them from receiving stem cell-based therapies. All the growth factors, proteins, extracellular matrix mediators, and other trophic cytokines collectively secreted by a stem cell are known as its secretome. Recently, secretome has shown promise for the treatment of glaucoma [13] and a plethora of other diseases [14, 15]. In this chapter, we describe detailed methods for secretome harvesting from TMSCs and secretome protein isolation. We also describe the quality control assays which should be taken into consideration so that secretome with utmost purity and quality can be isolated for successful downstream applications. Periocular injection of stem cell secretome into mice for exploring TM regeneration and glaucoma treatment is also described in this chapter.

2. Materials

2.1. Isolation and Explant Culture of TMSC from Human Tissue

  1. Donated human corneal tissues.

  2. DMEM/F12 medium supplemented with 50 μg/mL gentamicin, 1.25 μg/mL amphotericin B, 100 μg/mL streptomycin, 100 IU/mL penicillin (GASP) for corneal tissue sterilization.

  3. Dissection microscope.

  4. Surgical tools: No. 11 scalpel blades, scissors, forceps.

  5. For cell dissociation: TrypLE express enzyme or 0.25% trypsin.

  6. Culture ware: dishes and flasks of different volumes ranging from 10, 25 to 75 cm2.

  7. Fibronectin collagen (FNC) Coating solution for proper adherence of TM tissue explants/cells.

  8. 15 mL sterile conical tubes.

  9. Cell Strainer-70 μm nylon mesh.

  10. TMSC culture medium composed of multipurpose reduced-serum medium (OptiMEM-1) supplemented with 5% fetal bovine serum (FBS), 100 μg/mL streptomycin, 100 IU/mL penicillin, 50 μg/mL gentamicin, 10 ng/mL EGF (epithelial growth factor), 0.08% chondroitin sulfate, 200 μg/mL calcium chloride, 20 μg/mL ascorbic acid, 100 μg/mL bovine pituitary extract.

  11. Cell counter.

  12. A cell culture incubator maintained in sterile conditions at 5% CO2 and 37 °C temperature.

  13. Centrifuge.

  14. Phosphate buffered saline (PBS).

  15. Antibodies targeting CD90, CD105, CD73, and CD166, each conjugated with a distinct fluorescent color.

  16. Flow cytometer or access to a flow cytometer capable of reading fluorochromes in the excitation/emission range of FITC/PE/APC/BV510/PerCP, etc., depending on the fluorochrome used to tag the antibodies.

2.2. TMSC-Derived Trophic Factor (Secretome) Harvesting and Protein Isolation

  1. 1X PBS.

  2. Centricon devices.

  3. 50 mL sterile tubes.

  4. A centrifuge capable of rotating 50 mL tubes up to 4000× g for multiple hours at 4 °C.

  5. Ice bucket.

  6. Methanol.

  7. Chloroform.

  8. Distilled water.

  9. Protease inhibitor cocktail.

  10. Cell aspirator.

  11. Mini-centrifuge capable of rotating 1.5 mL tubes at 4 °C.

  12. Pipette tips, low binding.

2.3. Flow Cytometry Determination of TMSC Health Post-Secretome Harvesting

  1. TMSC culture medium.

  2. 1X PBS.

  3. TrypLE express system or 0.25% trypsin.

  4. Disposable pipettes.

  5. Annexin V binding buffer (commercially available).

  6. Fluorescent conjugated Annexin V.

  7. 7-AAD (7-Aminoactinomycin D).

  8. Flow cytometer capable of reading excitation/emission wavelengths in the range of FITC/PE/APC, etc., depending on the fluorochrome conjugated to Annexin V and 7-AAD wavelength.

2.4. Fluorescence Microscopy-Based Health Determination of TMSC Post-Secretome Harvesting

  1. TMSC culture medium.

  2. T-75 culture flasks.

  3. 1X PBS.

  4. TrypLE express system or 0.25% trypsin.

  5. Calcein AM: dissolve 50 μg/vial in 50 μL DMSO, store at −20 °C, use at 5 μg/mL.

  6. Propidium Iodide (PI), use at 2 μg/mL.

  7. Incubator.

  8. Fluorescent microscope capable of reading excitation/emission wavelengths in the range of DAPI/FITC/PE/APC.

2.5. Periocular Injection of TMSC Secretome

  1. Secretome from TMSC: secretome should be collected at log-phase growth condition (70–80% confluence).

  2. Hamilton syringes.

  3. Eye drops: 0.5% Proparacaine hydrochloride, Gentamicin, or Tobramycin eye drops.

  4. Lubricant: 2.5% Gonak hypromellose ophthalmic solution.

  5. Stereomicroscope with a movable stage, paddle, and a light source.

  6. Tonometer for intraocular pressure (IOP) measurement.

  7. Heating pad.

  8. Ice.

3. Methods

3.1. Isolation and Culture of TMSC from Human Tissue

  1. Wash human corneal tissues or corneal rims thoroughly (see Note 1).

  2. Immerse corneal tissues in DMEM/F12 with antibiotics GASP for 5 min (see Note 2). Repeat three times.

  3. Use a dissecting microscope to carefully dissect the TM tissue including the insert region [16, 17]. In parallel, prepare T-25 flasks with FNC coating (see Note 3).

  4. Explant culture: Put the TM tissue explant in a FNC-coated T-25 flask, add 1.5 mL TMSC culture medium, and incubate in a 37 °C and 5% CO2 cell culture incubator. Do not disturb it for the next 7 days so that cells can migrate from the attached TM tissue explant without disturbance.

  5. Replenish with fresh medium after 7 days and keep changing medium partially every 3 days for 2–3 weeks. The cultured cells will be trypsinized using TrypLE or trypsin, seeded into new flasks at a seeding density about 1000–2000 cells/cm2. Characterize the cells as in step 8.

  6. Single cell culture: Continuing from step 3, digest the TM tissue in a 15-mL with 1–2 mL TrypLE or trypsin in an incubator for 20–30 min, vortex every 10 min. Pipette the digested tissue gently to dissociate cells as single cells. Add same volume of DMEM/F12 with 2% FBS to terminate the digestion. Filter through a 70-μm nylon mesh to get rid of the tissue. Wash the cells with DMEM/F12 to completely remove TrypLE or trypsin.

  7. Count cell number and seed cells at 2000 cells/cm2 with stem cell culture medium into a suitable tissue culture dish or flask pre-coated with FNC. Passage cells when they are ~80–90% confluence and seed them at 1000–2000 cells/cm2.

  8. Characterize the cultured cells by explant culture or single cell culture for stem cell markers like CD90, CD73, CD166, CD105, etc., by flow cytometry [5, 18] and then used at passage 3 onwards for secretome harvesting. Cultured cells with more than 90% of whole cell population expressing these cell surface markers will be used for secretome harvesting.

3.2. TMSC-Derived Trophic Factor (Secretome) Harvesting and Protein Isolation

3.2.1. Harvesting Secretome from TMSC

  1. Culture cells in TMSC culture medium (containing supplements) as in Subheading 3.1. When the cells are in log-phase at ~70–80% confluence, prepare for secretome incubation.

  2. Remove the culture medium, wash the TMSC 4 times with 1X PBS to remove any FBS and growth factor remnants which are in the culture medium (see Note 4).

  3. Incubate TMSC with sufficient volume of basal medium that is devoid of any growth factors and supplements for 48 h (see Note 5).

  4. After 48 h, collect culture media in cold tubes that are already maintained on ice (see Note 6).

  5. Centrifuge the conditioned media at 2000× g for 5 min to remove any cell debris. Transfer the supernatant in fresh tubes, which is considered the secretome from this point forward. Assess the health of the remaining TMSC (step 6) and handle the secretome (steps 7 and 8) differently.

  6. According to the criteria for which cells must be qualified for healthy secretome harvesting, cells should show less than 5% cell death after secretome incubation in basal medium and harvesting [19]. The cells can be processed for flow cytometry by staining with Annexin-Vand 7-AAD (see Subheading 3.3) or can also be simply stained with Calcein AM and Propidium Iodide (PI) and do microscopy to show the cell viability (see Subheading 3.4). Viability can be assessed between centrifugation steps.

  7. Using 3KDa molecular weight cut off (3KDa MWCO) protein concentrator devices (Centricon), concentrate the secretome to 25× (e.g., initial 50 mL to 2 mL) or at a designated concentration. The time of centrifugation may vary depending on how fast the Centricon concentrators can filter. Usually, it should take 30 min to 1 h at 4000× g if Centricon devices are new, but longer if the device gets old (see Note 7).

  8. Collect and aliquot the concentrated secretome (supernatant) in 1.5-mL tubes at a designated volume (such as 200 μL/tube) and store the secretome at −80 °C for later use. Discard the flow through (see Note 8).

3.2.2. Protein Isolation from Concentrated Secretome for Proteomic Analysis

Proteins can be isolated from secretome using chloroform methanol method:

Based on our experience, this is the best method to isolate proteins from secretome because other methods using detergents can interfere with LC-MS/MS. This method will give dry protein material, free of salts and detergents, which performs well during critical steps like reduction, alkylation, etc. This method is adapted from Wessel and Flügge [20] with modifications as per our own experience with secretome.

Assuming a starting 2 mL of secretome at this stage:

  1. To 2 mL of secretome, add 8 mL (4 parts) of methanol and vortex thoroughly (see Note 9).

  2. Add 2 mL chloroform (1 part) and vortex thoroughly.

  3. Add 6 mL (3 parts) sterile double distilled water (ddH2O) and vortex. The mixture should become cloudy with precipitated protein flakes.

  4. Centrifuge for 1–2 min at 14,000× g which will result in three layers: a large aqueous layer on top, a circular flake of protein in the interphase, and a smaller chloroform layer at the bottom (see Note 10).

  5. Remove top aqueous layer carefully, trying not to disturb the protein flake (see Note 11).

  6. Add 8 mL (4 parts) methanol and vortex (maintain on ice again).

  7. Centrifuge 5 min at 20,000× g, which will slam dandruffy precipitate against the tube wall (see Note 12).

  8. Remove as much methanol as possible. Be careful, because the pellet is delicate. Remove all but a few μL of methanol with care (see Note 13).

  9. Dry the protein pellet (see Note 14).

  10. Take a small aliquot for measuring protein concentration using BCA assay (see Note 15).

  11. Perform LC-MS/MS if protein characterization is needed by proteomic analysis.

3.3. Flow Cytometry Determination of TMSC Health Post-Secretome Harvesting

TMSCs need to be characterized for their health post-secretome harvesting and a secretome should be used only from those cells which show <5% cell death. The cell health can be evaluated using Annexin-V and 7-AAD (7-Aminoactinomycin D) staining.

  1. Trypsinize TMSC (from which secretome has just been harvested, Subheading 3.2.1, step 5) using TrypLE for 5 min after 1X PBS wash and collect the cultured cells in 15 mL tubes.

  2. Wash the cells with 1X PBS again to remove any remaining TrypLE or medium.

  3. Use an Annexin VApoptosis Detection Kit containing Annexin V Binding Buffer and 7-AAD. Annexin V can be conjugated with FITC or APC which can be distinguished from 7-AAD channel.

  4. Make 1X solution of Annexin V Binding Buffer and wash cells with it to reduce background.

  5. Resuspend TMSCs at a concentration of 1 × 106 cells/mL in 1X Annexin V Binding Buffer and divide the cells into 6 different tubes with equal volume/equal cell number, 3 tubes for technical staining repeats and 3 tubes for technical unstained control.

  6. Pipette the cells gently and add 3–5 μL Annexin V to each allocated TMSC tube.

  7. Add 5 μL of 7-AAD to each TMSC tube.

  8. Incubate at room temperature in dark for 15–30 min.

  9. Centrifuge the cells at 300× g for 5 min and discard the supernatant.

  10. Resuspend the cells in 200 μL Annexin V Binding Buffer and analyze using a flow cytometry. Use unstained cells as control to compensate for background fluorescence.

  11. Viable cells do not stain with either Annexin Vor 7-AAD. Early apoptotic cells stain Annexin V but not 7-AAD. Apoptotic death or necrotic cells stain for both Annexin V and 7-AAD. Healthy TMSCs should have <5% of cells stain for either Annexin V or 7-AAD or both.

3.4. Fluorescence Microscopy-Based Health Determination of TMSC Post-Secretome Harvesting

Calcein AM and PI can also be used to evaluate cell viability after the secretome is collected. Healthy live cells will take up Calcein AM, a cell-permeant dye, but not PI. PI cannot penetrate intact membranes of live cells and can only be taken up by dead cells whose membrane integrity has been compromised.

  1. Use adherent TMSCs from which secretome has been harvested for this (see Subheading 3.2.1, step 5).

  2. Add appropriate basal medium devoid of serum and growth factor to TMSC flasks after taking the cultured basal media for secretome.

  3. Add Calcein AM at a final concentration as 5 μg/mL.

  4. Add PI at a final concentration as 2 μg/mL.

  5. Incubate for 30 min in dark in a cell culture incubator (see Note 16).

  6. Wash the cells with 1X PBS, twice.

  7. Add fresh 1XPBS or serum-free medium into the flask with stained cells.

  8. Image the cells under a fluorescent microscope capable of reading FITC/PE excitation/emissions spectra.

  9. Take pictures from at least 5 fields per sample and count the positively stained cells using ImageJ and calculate the percent-age of PI positive cells among the whole cell population (PI positive + Calcein AM positive cells). It should be less than 5%.

3.5. Periocular Injection of TMSC Secretome

The concentrated secretome prepared above (see Subheading 3.2.1, step 8) or isolated proteins from secretome (see Subheading 3.2.2, step 9), after dissolving in 1X PBS, can be used for periocular injection in mouse glaucoma models. After removing secretome from −80 °C freezer, it should be maintained on ice at all times and allowed to thaw only on ice. It should be brought to room temperature immediately before injection.

  1. Anesthetize mice using ketamine/xylazine (see Note 17).

  2. Measure IOP using a tonometer for rodents. IOP should be measured immediately after a mouse is not moving after anesthesia since anesthesia can reduce IOP. IOP measurement should be always performed at the same time of a day for consistency, since IOP fluctuates over the course of the day.

  3. Put the animal on the stage of a stereomicroscope with moving paddle and turn on the lamp to properly illuminate the eye.

  4. Apply one drop of 0.5% proparacaine hydrochloride on top of the eye to locally anesthetize the eye.

  5. Using a 33-gauge needle connected to a 25- μL Hamilton syringe, fill the syringe with 20 μL of 25× concentrated secretome and remove any air bubbles.

  6. Penetrate the needle through superior or inferior conjunctival fornix and then carefully insert the needle along the surface of the back of the eye. Hold the syringe steadily and slowly inject the 20 μL secretome into the periocular space. During injection, the injected secretome can be seen around the eye beneath the conjunctiva.

  7. Pull off the needle and add a drop of 2.5% Gonak hypromellose to protect the cornea (see Note 18).

  8. Optional: Periocularly inject secretome proteins dissolved in 1X PBS at an optimized concentration.

  9. Control medium (for secretome) or 1X PBS (for secretome proteins) should be injected as sham controls.

  10. Since periocular injection is minimally invasive and doesn’t affect the vision immediately after injection, both eyes can be injected if local IACUC approves. If injecting both eyes, the same treatment should be used on both eyes. If injecting only one eye, the other eye can serve as an untreated control.

  11. After injection, place the mice on a heat pad until awakening.

  12. IOP should be measured regularly. After a designated observation period, the effects on TM regeneration should be explored, such as outflow facility changes, TM cellularity and extracellular matrix changes, RGC function, etc. [6].

4. Notes

  1. Tissue handling should be performed in a certified Biosafety Level 2 (BSL2) cell culture hood using proper protective gear. Serological testing must be performed for each donor to exclude the possibility of contamination with HIV and hepatitis. A written informed consent must be obtained from the donor/donor family stating that corneal tissue is authorized to be used for research or for both clinical and research purposes (such that the corneal rims can be used for cell culture after removing the central button for corneal transplantation).

  2. TMSC number is reduced with age [21], hence corneas from younger donors will be likely to have more TMSCs and a higher rate of success in isolating TMSCs.

  3. FNC coating mix doesn’t need to be dried out before seeding cells or tissue explants.

  4. Don’t perform longer PBS incubations since they can cause cell loss via de-adherence and floating of the cells. Repeat this step 4 times to thoroughly remove all growth factors and FBS.

  5. Basal media for secretome shouldn’t contain any growth factors, additives, or FBS. The basal medium is the normal culture medium without any supplements. Since TMSCs are being cultured in reduced-serum OptiMEM-1, 5% fetal bovine serum (FBS), 10 ng/mL EGF (epithelial growth factor), 100 μg/mL bovine pituitary extract, 20 μg/mL ascorbic acid, 200 μg/mL calcium chloride, 0.08% chondroitin sulfate, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 50 μg/mL gentamicin, etc., the secretome incubation will be given only in OptiMEM-1 without anything else. Incubate TMSC in 10–12 mL basal medium/T75 flask for 48 h.

  6. From here the medium should be maintained in cold conditions on ice. All centrifugations will take place at 4 °C. Don’t touch samples or tubes with bare hands at any step because that might result in contamination of samples which will interfere with protein analysis. Use gloves during all steps. Keep samples covered.

  7. Don’t use higher speed centrifugations, which could result in protein loss.

  8. Sometimes, protein can precipitate on the membrane. Use a 1 mL pipette to triturate a few times and collect supernatant above the membrane carefully. Store the secretome in small aliquots, if intended for animal injection.

  9. Samples should be maintained on ice before and after vortexing.

  10. Remove tubes gently from centrifuge and not to disturb three layers. The tubes will go in a separate tube stand at this point which is maintained at room temperature.

  11. This is a very important step. At this step, a total of 18 mL liquid are in one tube. Allow the tubes to sit for 1–2 min to allow the layers to separate more clearly. Now very carefully, remove the aqueous layer by using a serological pipette or 1 mL pipette. There should be oil-like white droplets at around the 3–5 mL mark of the 50 mL tube. Remove the aqueous layer till that point. Be careful not to disturb or suck away the protein layer which is the white oil droplet-type material.

  12. Remove the tubes very carefully at this stage as mishandling can disturb the protein pellet from the wall. Banging the pellet will disturb it, if that happens, centrifuge again.

  13. If methanol removal is not accurate, some protein might also get removed with methanol. In that case, this methanol can be collected in a separate 1.5 mL tube and centrifuged again at 1500× g for 5 min and lost protein can be recovered in the form of a pellet. The protein concentration can be measured using BCA method.

  14. Don’t let the protein pellet overdry because it will be hard to solubilize it if is overdried. The trick is to add 1X PBS within 10–20 s after removing methanol and the protein pellet will dissolve nicely in PBS. The protein pellet can be dissolved in any desired volume of PBS depending on the pellet size. Make sure protein is dissolved nicely in PBS. Pipetting can be done to dissolve the pellet nicely, using low binding tips, otherwise it will lead to protein loss due to sticking of protein to pipette tips.

  15. Bradford or any other protein estimation assays are not ideal for secretome quantification; BCA works best. Store the rest of protein at −80 °C (Make 100–200uL aliquots depending on the application. Some of the aliquoted proteins can be saved for future downstream applications like western blot. Avoid freezing and thawing repeatedly). Aliquots can also be generated to provide a specific amount of proteins for mouse periocular injections.

  16. Longer incubation of cells with Calcein AM and PI can result into false positive staining and also detachment of the cells.

  17. Using keramine/xylazine needs the IACUC approval. For ketamine, a DEA license is needed, and double locked storage is necessary/required.

  18. During each injection of each eye, place the secretome tube back on ice. After injections for one experiment, discard any leftover secretome and don’t reuse secretome on another day as trophic factors such as RNA, microRNA, etc., will be degraded.

Acknowledgements

The work was supported by National Institutes of Health Grant EY025643 to YD and Start-up funding of Department of Ophthalmology, University of South Florida.

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