Human normal corneal epithelial cell and application thereof

Human normal corneal epithelial cell and application thereof

  • CN 106,591,216 B
  • Filed: 12/12/2016
  • Issued: 07/27/2021
  • Est. Priority Date: N/A
  • Status: Active Grant
First Claim
Patent Images

1. A human normal corneal epithelial cell is characterized in that the human normal corneal epithelial cell is classified and named as human normal corneal epithelial cell HNCEC/HL-008 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO:

  • C201550;

    the human normal corneal epithelial cells are primarily isolated and cultured from human normal corneal tissues, no foreign gene is introduced into the human normal corneal epithelial cells, and the human normal corneal epithelial cells are identified as human normal diploid cells through karyotype analysis;

    the morphology of the human normal corneal epithelial cells observed under a microscope is compact in arrangement, clear in cell boundary, strong in stereoscopic impression and polygonal epithelial cells, the morphology of the epithelial cells is still in a proliferation state and normally grows after 65 days of culture, the epithelial cells have a normal differentiation function under a three-dimensional culture condition and have no abnormal proliferation and tumorigenicity in vitro, and the primary isolated culture of the human normal corneal epithelial cells comprises the following steps;

    collecting pterygium-surgically excised pterygium-adjacent normal limbal tissue samples with informed consent of the patient or patient guardian;

    washing the separated tissue sample with 95-100% (v/v) ethanol, washing with PBS for 2 times, placing the tissue sample into a sterile culture dish containing precooled PBS, and removing residual fat in the tissue sample with dissecting forceps and scissors under a dissecting microscope;

    digesting a tissue sample by using a digestive fluid, wherein the digestive fluid is an HL medium containing collagenase and dispase, the HL medium is a DMEM and serum-free medium which are mixed according to a volume ratio of 1;

    3, and 5% (v/v) of FBS, 0.4 mu g/mL of cortisol, 5 mu g/mL of insulin, 8.4ng/mL of cholera toxin, 10ng/mL of epidermal growth factor, 24 mu g/mL of adenine, 100U/mL of penicillin, 100 mu g/mL of streptomycin, 0.25 mu g/mL of amphotericin B and 30 mu M of fasudil are also added;

    centrifuging the digested tissue to remove supernatant, and suspending the cell precipitate in pancreatin-EDTA with the mass volume ratio of 0.25% for digestion;

    adding DMEM medium containing 10% (v/v) FBS, and centrifuging to remove supernatant;

    adding dispase and DNase I in a warm water bath, and repeatedly blowing and beating a sample by using a gun head;

    adding a DMEM medium containing 10% (v/v) FBS, filtering the cell suspension by using a filter with the pore size of 40-70 mu m, collecting the filtered cell suspension, and centrifuging to remove the supernatant;

    the heavy suspension cell sediment is placed in HL culture medium and inoculated in a culture flask for culture, so as to obtain the normal corneal epithelial cells of human;

    the subculturing method of the human normal corneal epithelial cells comprises the following steps;

    when the human normal corneal epithelial cells proliferate to 70-90% abundance, washing the cells with 1 ×

    PBS, and digesting the monolayer cells with pancreatin-EDTA with the mass-volume ratio of 0.05%;

    adding DMEM to neutralize digestion reaction;

    centrifuging to remove supernatant, resuspending the cell pellet with HL medium, and inoculating to a culture flask for culture.

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