This protocol provides an advantageous strategy for enriching lacrimal gland stem cells for the regeneration and reconstruction of lacrimal gland. Lacrimal gland stem cells show long-term expansion capacity and the potential of differentiation. The main advantage of this technique is that the culture medium for lacrimal gland stem cells is serum-free, which shows the enormous value for lacrimal gland repair Begin by obtaining a euthanized six-to eight-week-old BALB/c male mouse and cutting the skin behind the ear to expose the lacrimal gland and the connective tissue around it.
Peel off the connective tissue by blunt dissection with the help of tweezers and remove the lacrimal gland. Immerse the lacrimal glands in a six-centimeter dish with four milliliters of 75%ethanol for 10 seconds and immediately rinse with 10-millimolar PBS solution twice. Cut the lacrimal glands into small fragments of about one millimeter cubed.
Transfer them to a sterile 15-milliliter centrifuge tube and treat the tissues with 500 microliters of 25 units per milliliter dispase and 500 microliters of 0.1%collagenase I for one hour at 37 degrees Celsius. Treat the lacrimal gland fragments with one milliliter of 0.05%trypsin EDTA for 10 minutes at 37 degrees Celsius and dissociate the fragments into single cells by pipetting repeatedly. Filter the suspension through a 70-micrometer filter, collect the filter into a sterile 15-milliliter centrifuge tube, and centrifuge at 150 times G for five minutes.
After centrifugation, remove the supernatant, add 10 milliliters of 10-millimolar PBS solution to wash the cell pellet, and centrifuge the suspension at 150 times G for five minutes. Repeat the washing of the cell pellet. Then, remove the supernatant and add one milliliter of a 1:1 ratio of Dulbecco's Modified Eagles Medium and Ham's F-12 to resuspend the cell pellets.
Add 20 microliters of matrix gel lacrimal gland stem cell medium matrix at the center of the well of a 24-well plate. Use a pipette tip to expand it to a circle of about six-to eight-millimeter diameter and incubate the mixture for 20 minutes at 37 degrees Celsius to pre-coat the well. Use a cell counter to determine the number of cells and add 40 microliters of lacrimal gland stem cell medium, or LGSCM, with a total of 10, 000 cells into 40 microliters of the matrix gel.
Mix them gently with a pipette. Use a pipette to carefully drop 80 microliters of the mixture over the pre-coated area in each well of the 24-well plate. Incubate the mixture for 20 minutes at 37 degrees Celsius and add 600 microliters of LGSCM.
Change the culture medium in each well once every two days. After seven days of culture, look for LGSC spheres that are 100 to 300 micrometers in diameter under the inverted microscope. Use this protocol to isolate and culture primary lacrimal gland stem cells of Rosa26 and NOD mice.
After seven days of culture, remove the culture medium from the sphere culture well. Disaggregate the LGSC spheres by incubation in 20 microliters of 10 units per milliliter dispase and 100 microliters of 10-millimolar PBS for 30 minutes at 37 degrees Celsius. Transfer the suspension to a 15-milliliter centrifuge tube and centrifuge at 150 times G for four minutes.
Remove the supernatant. Treat the spheres with one milliliter of 0.05%trypsin EDTA for five minutes at 37 degrees Celsius Add one milliliter of 0.05%trypsin inhibitor and pipette repeatedly to neutralize the trypsin and dissociate the spheres into single cells. Centrifuge at 150 times G for five minutes and remove the supernatant to pellet the LGSCs.
Add one milliliter of LGSCM to resuspend the lacrimal gland stem cell pellet and plate the cells as previously demonstrated. If performing procedure one, extend the culture time of the LGSCs from seven to 14 days with the lacrimal gland stem cell culture system for random differentiation. For procedure two, change the ratio of the matrix gel to LGSCM from 1:1 to 1:2 at the beginning of the lacrimal gland stem cells culture for the differentiation of ductal cells.
Seed the lacrimal gland stem cells into the matrix for induction for 14 days. For procedure three, after passage, replace the LGSCM with LGSCM 10%FBS for differentiation of acinar cells. Induce differentiation for 14 days.
After one week of culture, Kr14 and Ki67 were expressed in all spheres formed by lacrimal gland stem cells. The spheres reached a diameter of 100 micrometers. Hematoxylin and eosin staining showed cellularity at day seven.
The enrichment factors of lacrimal gland stem cells obtained after seven days of primary culture and subculture indicated that the cells enriched by this method had a strong proliferative ability. In this system, lacrimal gland stem cells could be passaged over 40 times and still maintain stem cell characteristics. Lacrimal gland stem cells were induced to form more buds by fetal bovine serum and a low proportion of matrix gel.
Hematoxylin and eosin staining indicated that fetal bovine serum could induce the spheres to produce more cavitating structures. After orthotopic injection of Rosa lacrimal gland stem cells in NOD mice, new lacrimal lobules were formed adjacent to the lacrimal glands. Most of the lobules were composed of mature acinar cells with high expression of AQP5 and there was intralobular duct formation with low AQP5 expression.
The amount of tear secretion on the Rosa lacrimal gland stem cells injection side was higher than on the control side, but lower than in the wild-type mice The matrix gel and its related mixture must always be kept on ice before use and the microcentrifuge tube and tips in contact with matrix gel should be pre-cooled. The system provides an ideal model for further study of lacrimal gland stem cells, lacrimal gland repair, and drug screening for lacrimal gland diseases.
