Corneal interstitial cells, known as keratocytes, exhibit a resting phenotype characterized by a distinctive dendritic morphology and a very low to near absent proliferation rate in vivo. At the time of acute injury, keratocytes begin to mitotically divide, assume a fibroblast morphology, and migrate to the area of injury. In vitro, primary keratocytes were able to maintain a quiescent state in serum-free or low mitogen serum medium and were similar to quiescent keratocytes in vivo in terms of cell morphology and stromal secretory effects. Fetal bovine serum was able to proliferate keratocytes, but was also able to change keratocytes into fibroblasts or myofibroblasts. Source: Human Stem Cell Culture
Operation method
Isolation of primary human corneal mesenchymal stromal cells
Materials and Instruments
Intact human corneas Move (a) The cornea was cleaned with CMF-SalineG for 3 × 5 min. (b) The residual sclera, conjunctiva and iris were clipped. (c) Add 2 ml of 1.2 U neutral protease II and shake overnight at 4°C on a shaker. (d) The corneas to which neutral protease II was added were shaken at 4°C for 30 min. (e) Corneas were washed with DMEM/F-12/GASP. (f) Under a dissecting microscope, epithelial and endothelial cells were carefully removed. (g) The epithelial and endothelial cell surfaces of the basement membrane were gently scraped with a plastic scraper. Observe under a microscope to ensure complete removal of these cell layers. (h) The corneal stroma was washed once with fresh medium. (i)The stroma was cut into small pieces of about 2 mm using a scalpel or safety blade or fine surgical scissors. (j) The stroma was digested with collagenase prepared with DMEM/F-12/GASP at 37°C for 3 h until most of the tissue disappeared. (k) Centrifuge at 400 g for 10 min, and discard the supernatant. (l) Cells were resuspended with DMEM/F-12/GASP and treated with 70 μm cell strainer to filter the digest and centrifuge. (m) Repeat the above washing steps two times, and the cells were counted after each time. (n) Isolated mesenchymal cells were resuspended and inoculated in plastic tissue culture dishes at a density of 1 × 104cm2 using MJM. (o) The medium was changed every 3 days. (P) Transfections were digested with trypsin when cells reached 90% confluence: the medium was aspirated and washed once with CMF-SalineG. Add human trypsin or TrypLE to just cover the cells, 37°C, 10 min. add DMEM/F-12/2FB to terminate digestion. Cell counting. Centrifuge the resuspended cells and discard the supernatant. Cells were resuspended with sufficient amount of freshly prepared MJM medium and inoculated at a density of 1×104cm2. For more product details, please visit Aladdin Scientific website.
CMF-SaIineG Neutral Protease II L-type collagenase 1mg ml in DMEM F-12 GASP CMF-SaIineG formulated with TrypLEExpress or 0.25% Membrane Protease DMEM F-12 GASP DMEM F-12 2FB GASP:DMEM F-12 GASP with 2% FBS CMF GASP
3.5cm plastic tissue culture dish or 6-well plate Scalpel or single-edged safety blade Cell strainer 70μm blood nylon filter Plastic scraper "CellLifter" or "CellScraper" Curved iris shears 11cm(4 -3 8in.) Jeweler's forceps 10cm(4in.) Corneal shears 19mm blade Pointed Colibri suture clamp 0.1mm