Materials and Reagents

Micropipette tips (0.5-20 µl, 100-200 µl, 1,000 µl) (Greiner Bio-One)
60 mm cell culture dish (Corning, Falcon^®, catalog number: 353004 )
100 mm cell culture dish (Corning, Falcon^®, catalog number: 353003 )
Syringe filter 0.2 μm (VWR, catalog number: 28145-501 )
Disposable Scalpel blades No. 10 (pfm Medical ag, Feather^®, catalog number: 201000010 )
Serological pipettes (5 ml, 10 ml) (Corning, Stripette^TM)
15 ml conical tubes (Greiner Bio-One, catalog number:188271)
T75 flasks (Corning, catalog number: CLS430641 )
Reversible cell strainers (Stem Cell Technologies, catalog number:27215)
Cell filter 20 µm (Sysmex Partec GmbH, Görlitz, Germany)
Collagenase A (Sigma-Aldrich, Roche Diagnostics, catalog number: 10103578001 )
Dulbecco’s Phosphate buffered saline (DPBS) (Pan Biotech, catalog number: P04-361000 )
0.25% Trypsin-EDTA (Thermo Fisher Scientific, Gibco^®, catalog number: 25200056 )
0.05% Trypsin-EDTA (Thermo Fisher Scientific, Gibco^®, catalog number: 25300054 )
Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Thermo Fisher Scientific, Gibco^TM, catalog number: 11960044 )
Fetal Bovine Serum (FBS) (Thermo Scientific, Gibco^TM, catalog number: 10082147 )
Penicillin-Streptomycin/Amphotericin B Mixture (Pan Biotech, catalog number: P06-07300 )
Keratinocyte serum-free medium (KSFM) (Life Technologies, catalog number: 10724-011 )
Keratinocyte supplements, contains bovine pituitary extract, human recombinant EGF (Life Technologies, catalog number: 37000-015 )
iMatrix 511(Nippi, catalog number: 892012 )
70% Ethanol
Cytokeratin (CK) 15 (Invitrogen, catalog number: MA5-11344 )
p63α (Abcam, catalog number: ab32353 )
CCAAT/enhancer-binding protein delta (Abcam, catalog number: ab139730 )
CK19 (Abcam, catalog number: ab52625 )
CK3 (Invitrogen, catalog number: MA1-5763 )
CK12 (Abcam, catalog number: ab185627 )
Connexin-43 (Abcam, catalog number: ab235282 )
Collagenase solution (see Recipes)
KSFM complete medium (see Recipes)
Laminin coated plates (see Recipes)

Equipment

Pipette aid
Micropipette (P20, P200, P1000)
Microdissection Scissors
Forceps
Scissors
Hemocytometer
Biosafety cabinet
CO₂ incubator
Centrifuge
Phase contrast inverted microscope with a camera (Objectives 4x, 10x, 20x)
Freezer -20 °C
Refrigerator 2-8 °C

Software

CapturePro 2.10.0.1 (JENOPTIC Optical systems GmbH)

Procedure

Dissection of limbus
1. Organ-cultured corneoscleral tissue with appropriate research consent and ethics approval may be provided by a cornea bank if the corneas are not suitable for transplantation due to low corneal endothelial cell density (< 2,200 cells/mm²), or the presence of neurological disorders or malignancies in the donor. Donor cornea remnants after corneal endothelial transplant preparation are also a valuable source if appropriate research consent and approval have been obtained.
2. Place the corneoscleral tissue in a 60 mm culture dish and wash with DPBS twice. Cut the tissue into 12 three-clock hour sectors using a scalpel blade and forceps.
3. Make incisions at 1 mm before and beyond the anatomical limbus to get limbal segments (see Video 1).
  
  Video 1. Limbal Epithelial Progenitor Cell Isolation
Isolation of limbal epithelial progenitor cells
1. Place the limbal segments into a 60 mm dish containing 4 ml of collagenase A (2 mg/ml) and cut the limbal segments into smaller pieces with the scalpel blade. Incubate at 37 °C with 5% CO₂ overnight to digest the stromal collagen (get limbal epithelial cell clusters).
2. After overnight incubation, triturate several times with 1 ml pipette and observe for the presence of cell clusters and single cells in the microscope (Figure 2). The cell clusters are supposed to consist of limbal epithelial progenitor cells, stromal, and melanocyte niche cells.
  Note: In case of incomplete digestion of limbal segments after overnight incubation and trituration, re-incubate for 2 more hours in the same solution at 37 °C with 5% CO₂ for complete digestion. On the other hand, over digestion of tissue might adversely affect cell viability and the quality of cells.
  
  Figure 2. Isolation of limbal epithelial progenitor cells. A. The corneal scleral rim (left) was cut into sectors and each sector was trimmed off 1mm before and after the limbal region (right). B. Different sizes of limbal clusters and single cells (left) formed after overnight incubation of limbal segments in collagenase solution (x40 magnification). Limbal cluster-derived limbal epithelial cells cultured in KSFM media at 50% confluence (right, top) and 100% confluence (right, bottom) (x40 magnification).
3. Separate cell clusters from single cells by using reversible cell strainers with a pore size of 37 µm. The single cells pass through the cell strainer and the clusters are retained. Wash the strainers with PBS twice to remove single cells. Reverse the strainer and place it on a 15 ml Falcon tube or on a 60 mm dish. Add 0.25% trypsin-EDTA (5 ml) to flush clusters into petri dish or tube and incubate at 37 °C for 10-15 min to dissociate the clusters into single cells.
  Note: In place of reversible cell strainers, 20 µm cell filters can be used. In the video, we used 20 µm filters.
4. After incubation, triturate the cell suspension with a 1ml pipette. Inhibit trypsin digestion using 5ml of DMEM containing 10% FBS. Transfer the cell suspension into a 15 ml Falcon tube and centrifuge at 200 x g for 5 min.
5. After centrifugation, resuspend the cell pellet in 1 ml of KSFM complete media for feeder-free cultures. These cells can also be used for clonal assays on feeder layers.
Expansion of limbal epithelial progenitor cells
1. Seed the limbal cluster-derived single cells (1 x 10³ cells/cm²) onto LN511E8-coated plates (flasks or plates depending on cell number obtained. Preparation of laminin-coated plates described in Recipe 3.
  Note: Do not allow the laminin-coated flasks to dry.
2. Cultivate for 7 to 10 days at 37 °C with 5% CO₂ in KSFM complete medium to expand limbal epithelial cells. Change media every 2 days.
  Note: If any stromal fibroblast contamination occurs after 7 days in culture, remove the stromal fibroblasts, which tend to adhere less firmly to the LN matrix, by using 0.05% trypsin-EDTA. Remove the media and wash it with DPBS. Incubate with 4 ml of 0.05% trypsin-EDTA for 2 min. Wash the flasks with DMEM containing 10% FBS followed by one wash with KSFM. Add 12 ml of KSFM complete medium and continue with culture
3. To verify the expanded population for the presence of stem/progenitor cell population, various stem/progenitors markers (Cytokeratin (CK) 15, p63α, CCAAT/enhancer-binding protein delta, CK19) and differentiated markers (CK3, CK12, Connexin-43) can be used by means of Reverse Transcription Polymerase chain reaction and by immunocytochemistry.
Sub-cultivation of limbal epithelial progenitor cells
1. At 70 to 80% confluency, remove the media and wash once with DPBS.
2. Add 4 ml of trypsin-EDTA (0.05%) and incubate at 37 °C with 5% CO₂ for 5 min.
3. After incubation, add 4 ml of DMEM containing 10% FBS to inhibit trypsin action and mix well.
4. Transfer the cell suspension into 15 ml tube and centrifuge at 200 x g for 5 min. Dissolve the cell pellet in KSFM complete medium and use cells for the application of choice or for subculturing.
  Note: Over confluence (more than 80%) and prolonged trypsin digestion (more than 5 min) adversely affect cell viability and the quality of cells during sub-culturing. Always passage cells at 70 to 80% confluence. Avoid prolonged incubations in trypsin.

Data analysis

The conditions provided in this protocol have been optimized to isolate and expand LEPCs. A detailed analysis of the isolation and expansion of the limbal epithelial progenitor cells can be found at Polisetti et al., 2017.

Recipes

Collagenase solution
Reagent Collagenase A
Components and Preparation 5 mg Collagenase A
220 ml of DMEM High Glucose
25 ml of fetal calf serum
5 ml of Penicillin-Streptomycin/Amphotericin B Mix
Mix well by inverting
Method of Sterilization Sterile Filter with 0.2 µm filter
Notes Prepare 10 ml aliquots
Storage -20 °C
KSFM Media
Medium Keratinocyte Serum-Free Medium (KSFM) complete
Components and Preparation 500 ml of KSFM Basal Medium
25 mg of BPE (add entire amount)
2.5 µg of EGF (add entire amount)
5 ml of Penicillin-Streptomycin/Amphotericin B Mix
Mix well by inverting
Method of Sterilization None
Notes Prepare aliquots if needed
Protect from light
Storage 1 month at 2-8 °C
Laminin coated plates
1. Dilute 75 µl of iMatrix-511 in 8 ml of 1x DPBS
2. Coat the T75 flask with 8 ml of dilute iMatrix-511 (0.5 µg/cm²) solution for 1 h at 37 °C, 5% CO₂ in the incubator, or at 4 °C overnight
3. After incubation, remove the iMatrix-511 solution and add 10 ml of KSFM complete medium

Acknowledgments

This work is supported by DFG Grant INST 410/45-1 FUGG. This protocol was adapted from previous work (Polisetti et al., 2017).

Competing interests

The authors declare that they have no competing interests.

References

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