Human normal corneal epithelium cells and application thereof

A technology of corneal epithelial cells and normal cells, applied in the field of cell biology, to achieve the effect of strong three-dimensional sense, clear cell boundaries and tight arrangement

Active Publication Date: 2017-04-26
SHENZHEN EYE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At this stage, there has never been a "normal cell" that can be

Method used

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  • Human normal corneal epithelium cells and application thereof
  • Human normal corneal epithelium cells and application thereof
  • Human normal corneal epithelium cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] [Example 1] Primary isolation and culture of primary human normal corneal epithelial cells

[0047] (1) With the informed consent of the patient or patient guardian, collect the normal tissue samples next to the pterygium surgically removed from patients with pterygium.

[0048] (2) Preparation of digestive solution: HL medium containing 0.2 mg / mL of collagenase and dispase; among them, HL medium is: DMEM (GIBCO#11965-092) and serum-free medium SFM (GIBCO#10744- 019) mix by volume ratio 1:3, add the fetal calf serum of 5% (v / v) simultaneously, and 0.4 μ g / mL cortisol (hydrocortisone), 5 μ g / mL insulin (ins μ Lin), 8.4 ng / mL cholera toxin ( cholera toxin), 10ng / mL epithelial growth factor (EGF), 24μg / mL adenine, 100U / mL penicillin, 100μg / mL streptomycin, 0.25μg / mL amphoteric Mycin B (Fungizone), 30 μM Fasudil (Fasudil), the above medium needs to be filtered through a 0.22 μm pore size filter).

[0049] (3) Wash the isolated tissue sample once with 95-100% (v / v) ethanol...

Embodiment 2

[0058] [Example 2] Subculture of normal human corneal epithelial cells

[0059] (1) When the human normal corneal epithelial cells cultured in T25 or T75 culture flask proliferate to 70-90% abundance, wash the cells twice with 1×PBS (0.01M, pH 7.4), and then wash with 0.05% ( Mass volume ratio) Trypsin-EDTA digested monolayer cells for 2-5 minutes.

[0060] (2) Add 10 mL of complete DMEM to neutralize the digestion reaction for 1-2 minutes.

[0061] (3) Centrifuge at 1000rmp for 5 minutes, remove the supernatant, resuspend the cell pellet and inoculate in 10mL HL medium.

[0062] (4) If necessary, 1×10 6 The epithelial cells were resuspended in 1-2 mL of cell freezing solution (90% fetal bovine serum and 10% DMSO, v / v), and stored in liquid nitrogen for future use.

[0063] Subculture human normal corneal epithelial cells according to the above method, and the cell growth curve of the culture establishment line is as follows: figure 2 , continuous subculture for 65 days, ...

Embodiment 3

[0064] [Example 3] Karyotype analysis and identification of human normal corneal epithelial cells

[0065] (1) When human normal corneal epithelial cells (1×10 6 ) in the exponential growth phase, add colchicine at a final concentration of 0.2 μg / mL, and continue culturing for 3.5 hours.

[0066] (2) Beat the cells repeatedly to make them fall off, and centrifuge at 2000rpm for 5 minutes to harvest the cells.

[0067] (3) Discard the supernatant, add 8 mL of 0.075 mol / L KCl solution pre-warmed at 37°C, gently blow and beat the cell mass to mix, and place at 37°C for hypotonic treatment for 25 minutes.

[0068] (4) Add 1 mL of freshly prepared fixative (methanol: glacial acetic acid = 3:1, v / v), carefully pipette, mix, and centrifuge at 2000 rpm for 5 minutes.

[0069] (5) Discard the supernatant, add 8 mL of fixative, pipette to form a cell suspension, and fix at room temperature for 20 minutes.

[0070] (6) Centrifuge at 2000rpm for 5 minutes, discard the supernatant, and ...

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Abstract

The invention discloses human normal corneal epithelium cells and an application thereof. The cells are named as human normal corneal epithelium cells HNCEC/HL-008, and preservation number of the human normal corneal epithelium cells is CCTCC NO: 201550. The cell strain is prepared from a para-pterygium normal limbal tissue sample excised from an operation on a pterygium patient; based upon karyotype analysis, the cells are identified as human normal diploid cells; the cells, observed under a microscope, are represented as epithelial cells which are closely distributed, clear in cell boundary, strong in three-dimensional effect and polygonal; the cells can constantly maintain a uniform morphology for 65 days and can still achieve normal growth under a proliferative status, and the cells, under a three-dimensional culture condition, have a normal differentiation function. The human normal corneal epithelium cells are applicable to physiological research of human normal cells, drug toxicity research and detection of in-vitro normal cells as well as pathogenesis research of cornea and related cornea diseases including viral keratitis.

Description

technical field [0001] The invention belongs to the field of cell biology and relates to a human normal corneal epithelial cell and its application. Background technique [0002] Vital organs such as lung, kidney, liver, pancreas and skin are composed of organ-specifically differentiated epithelial cells. These specifically differentiated epithelial cells are directly related to the specific functions of different organs, such as the gas exchange function of the lungs, the filtering function of the kidneys, the detoxification and neutralization functions of the liver, the production of insulin by pancreatic cells, and the protection of the skin from the external environment. s damage. Once these vital organs become diseased or degenerate, human health will be threatened, because these vital organs are difficult to be replaced, and specific cells of different organs cannot replace cells of other organs. These specifically differentiated cells are difficult to regenerate, le...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/0625G01N33/5014G01N33/5044G01N2333/46G01N2800/16
Inventor 叶琳李晖王玲成洪波叶立娜丘建斌魏高斌王媛柯山
Owner SHENZHEN EYE HOSPITAL
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