METHOD FOR FACILITATING FUNCTIONS AND CHARACTERISTICS OF CORNEAL ENDOTHELIAL CELLS

Abstract

The present disclosure discloses a method for facilitating functions and characteristics of corneal endothelial cells, comprising the following steps of: separating and culturing human orbital adipose-derived stem cells, and extracting a conditioned culture medium; separating and culturing primary human corneal endothelial cells; adding the conditioned culture medium in a basal culture medium for the human corneal endothelial cells, and culturing and proliferating the human corneal endothelial cells. In the present disclosure, the human corneal endothelial cells cultured by the conditioned culture medium extracted from human orbital adipose-derived stem cells have high adherence and proliferation capacities. Human corneal endothelial cells cultured in vitro can be sub-cultured over 10 generations. The proliferation multiple is higher and the morphology and functions of the human corneal endothelial cells can be maintained. Experiments on animals have proved that the human corneal endothelial cells cultured in vitro have excellent cell repair effects.

Claims

1. A method for facilitating functions and characteristics of corneal endothelial cells, comprising following steps: separating and culturing human orbital adipose-derived stem cells, and extracting a conditioned culture medium; separating and culturing primary human corneal endothelial cells; and adding the conditioned culture medium in a basal culture medium for the human corneal endothelial cells, and culturing and proliferating the human corneal endothelial cells.

2. The method according to claim 1, wherein a method for separating and culturing human orbital adipose-derived stem cells comprises following steps: collecting human orbital adipose tissues under sterile conditions, washing for several times with PBS, soaking for 30 s with ethanol, washing for several times with PBS again, removing megascopic blood vessels and connective tissues, cutting into particles, adding collagenase digestion solution, and shaking and digesting in a constant-temperature shaker; then, adding a same volume of low-sugar DMEM culture medium containing FBS for neutralization, and centrifuging; discarding supernatant lipid and liquid, re-suspending with sterile PBS, centrifuging, discarding supernatant liquid, adding a DMEM culture medium, filtering with a filter screen, mixing uniformly and transferring to a sterile culture dish, and culturing in an incubator containing 5% CO.sub.2 at 37 C.; replacing the culture medium for the first time after 48 to 72 hours, subsequently every 2 to 3 days; and sub-culturing when the cell fusion reaches 80% to 90%.

3. The method according to claim 1, wherein a method for extracting a conditioned culture medium comprises following steps: using O-ASCs of the second to tenth generations, discarding the culture medium after the growth rate of O-ASCs reaches 50% to 80%, rinsing once with sterile PBS, adding a DMEN culture medium to continuously culture for 12 to 24 hours, collecting supernatant liquid in the cell culture medium, filtering the collected supernatant liquid by a filter to obtain a conditioned culture medium for human orbital adipose-derived stem cells, and storing at 80 C. for standby.

4. The method according to claim 1, wherein a method for separating and culturing primary human corneal endothelial cells comprises following steps of: microscopically tearing down the endothelium and Descemet's membrane of the cornea by a pair of forceps, incubating in a basal culture medium in an incubator at 37 C. overnight for stabilization; centrifuging, discarding supernatant liquid, and adding collagenase for digestion; and, separating corneal endothelial cells from the Descemet's membrane by pipetting for multiple times, centrifuging, and discarding the supernatant liquid to obtain primary human corneal endothelial cells.

5. The method according to claim 1, wherein a method for culturing and proliferating the human corneal endothelial cells comprises the following steps: re-suspending the obtained primary human corneal endothelial cells by a basal culture medium containing the conditioned culture medium, inoculating the cell suspension to a well of a culture plate, and culturing under 5% CO.sub.2 at 37 C.; replacing the culture medium for the first time after 48 hours, subsequently every other day; and sub-culturing at a ratio of 1:2 after the cells are fused.

6. Human corneal endothelial cells prepared by the method according to claim 1.

7. The human corneal endothelial cells according to claim 6, wherein the cells are polygonal, approximately hexagonal, and cells are densely joined and arranged in a single-layer mosaic pattern.

8. An application of the human corneal endothelial cells according to claim 6 in preparing medicines for treating decompensation of corneal endothelium.

9. A culture medium for culturing corneal endothelial cells according to claim 3, comprising a basal culture medium for human corneal endothelial cells and the conditioned culture medium having a mass percentage of 10% to 20%, wherein the basal culture medium for human corneal endothelial cells contains Opti-MEM-I, fetal bovine serum (FBS), epidermal growth factor (EGF), ascorbic acid, CaCl.sub.2, chondroitin sulfate and a mixed solution of penicillin and streptomycin.

10. The culture medium according to claim 9, wherein in the Opti-MEM-I, a volume percentage of FBS is 8%, a concentration of EGF is 5 ng/mL, a concentration of ascorbic acid is 20 g/mL, a concentration of CaCl.sub.2 is 200 mg/L, a weight per volume percentage of chondroitin sulfate is 0.08% (w/v), and a volume percentage of the mixed solution of penicillin and streptomycin is 1% to 1.25%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 shows the morphology of O-ASC cells and detection of cell-related markers by immunofluorescence by an inverted phase contrast microscope, where A and B show the separation and culturing of O-ASCs, and C shows immunofluorescence (vimentin).

[0042] FIG. 2 is a diagram showing the primary culture of Hcecs and the detection of markers thereof, where A to C show the primary culture of Hcecs, D shows the cell aging and deformation of Hcecs (P8), and E to G show detection of cell-related markers by immunofluorescence.

[0043] FIG. 3 shows a scratch test (for detecting the proliferation capacity of cells).

[0044] FIGS. 4.1 and 4.2 show detection of related makers of sub-cultured Hcecs, where FIG. 4.1 shows the detection by westernblotting and FIG. 4.2 shows the detection by immunofluorescence.

[0045] FIGS. 5.1 and 5.2 show the treatment of decompensation of animal corneal endothelium by human corneal endothelial cells cultured in vitro, where FIG. 5.1 shows the treatment of decompensation of rabbit corneal endothelium and FIG. 5.2 shows the treatment of decompensation of monkey corneal endothelium.

[0046] FIG. 6 shows living human corneal endothelial cells and human corneal endothelial cells cultured in vitro in the present disclosure.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0047] It is to be noted that the following detailed description is exemplary, only aimed at providing further understanding of the present disclosure. Unless otherwise specified, the technical and scientific terms used herein have meanings the same as the common meanings interpreted by those skilled in the art.

[0048] It is to be noted that the terms used herein are merely for describing specific implementations and not intended to limit the exemplary implementations of the present disclosure. As used herein, unless otherwise specified in the context, a singular form also includes a plural form. In addition, it should be understood that the term contain and/or include, when used in the description, means the presence of features, steps, operations and/or a combination thereof.

Explanation of Terms

[0049] PBS is the abbreviation of a phosphate buffer solution, which is a conventional buffer solution approximate to the physiological conditions of the human body.

[0050] The DMEM culture medium is a Dulbecco's modified Eagle's culture medium, including low-sugar DMEM and high-sugar DMEM. It is a conventional basal culture medium.

[0051] The Opti-MEM-I is a reduced serum culture medium. It is the modified form of the EMEM basal culture medium and is a medium formed by adding HEPES, sodium bicarbonate, hypoxanthine, thymine, sodium pyruvate, L-glutamine, insulin, transferrin and the like in the EMEM basal culture medium. This culture medium is a conventional reduced serum culture medium.

[0052] The materials and reagents used in the present disclosure can be obtained by conventional means. For example, the low-sugar DMEM culture medium is purchased from HyClone; the collagenase I is purchased from Sigma; the cell adherence reagent (FNCcoatingmix) is purchased from Usbio; the Opti-MEM-I is purchased from Gibco; and, the mixed solution of penicillin and streptomycin containing 10000 g/mL of penicillin and 10000 g/mL of streptomycin is purchased from Beijing Solarbio Science & Technology Co., Ltd.

Embodiment 1

[0053] 1. Separation and Culture of Human Orbital Adipose-Derived Stem Cells, and Extraction of a Conditioned Culture Medium

[0054] Separation and culture of O-ASCs: the adipose was from a patient who experienced blepharoplasty in the medical cosmetology department of Qilu Hospital. Human orbital adipose tissues were connected under sterile conditions; and under sterile conditions, the human orbital adipose tissues were washed for three times with PBS, soaked for 30 s with ethanol having a volume percentage of 75%, washed for three times with PBS again, removed with megascopic blood vessels and connective tissues, cut into particles in 1 mm.sup.3, added with 2 times in volume of 0.1% collagenase digestion solution I, and slowly shaken and digested for 1 hour in a constant-temperature shaker at 37 C. Then, a same volume of low-sugar DMEM culture medium containing 10% FBS was added for neutralization, and the mixture was centrifuged at 300g for 10 minutes. Supernatant lipid and liquid were discarded; the mixture was re-suspended with sterile PBS and centrifuged at 300g for 5 minutes; supernatant liquid was discarded; a proper amount of stem cell culture medium was added; and the mixture was filtered with a 100 m filter screen, mixed uniformly and transferred to a sterile culture dish, and cultured in an incubator containing 5% CO.sub.2 at 37 C. The culture medium was replaced for the first time after 48 to 72 hours and subsequently every 2 to 3 days. Sub-culturing or other experiments were performed when the cell fusion reaches 80% to 90%. The separated and cultured O-ASCs are shown in FIG. 1.

[0055] Extraction of the conditioned culture medium: O-ASCs of the second to tenth generations were used, and the culture medium was discarded after the growth rate of O-ASCs reaches 50% to 80%; the O-ASCs were rinsed once with sterile PBS and then added with a fresh stem cell culture medium to continuously culture for 12 to 24 hours; supernatant liquid in the cell culture medium was collected, and the collected supernatant liquid was filtered by a 0.22 m filter to obtain a conditioned culture medium for O-ASCs; and the conditioned culture medium was stored at 80 C. for standby.

[0056] The basal culture medium for human corneal endothelial cells contains Opti-MEM-I and is added with fetal bovine serum (FBS), epidermal growth factor (EGF), ascorbic acid, CaCl.sub.2, chondroitin sulfate and penicillin-streptomycin. In the Opti-MEM-I, the volume percentage of FBS is 8% (v/v), the concentration of EGF is 5 ng/mL, the concentration of ascorbic acid is 20 g/mL, the concentration of CaCl.sub.2 is 200 mg/L, the weight per volume percentage of chondroitin sulfate is 0.08% (w/v, g/100 mL), and the volume percentage of the mixed solution of penicillin and streptomycin is 1% (v/v).

[0057] The proportion of the conditioned culture medium is 10% to 20%.

[0058] 2. Primary Culture of Human Corneal Endothelial Cells (Hcecs)

[0059] The endothelium and Descemet's membrane of the cornea from the donor were torn down by a pair of forceps, and then incubated in a basal culture medium (Opti-MEM-I, 8% of FBS, 5 ng/mL of EGF, 20 g/mL of ascorbic acid, 200 mg/L of CaCl.sub.2, 0.08% of chondroitin sulfate and 1% of the mixed solution of penicillin and streptomycin) in an incubator at 37 C. overnight for stabilization. Then, centrifugation was performed at 300g for 5 minutes. Supernatant liquid was discarded, and 0.1% collagenase I (w/v, g/100 mL) was added for digestion for 1 to 2 hours at 37 C. Corneal endothelial cells were separated from the Descemet's membrane by pipetting for multiple times, and then centrifuged at 400g at 5 minutes. The supernatant liquid was discarded, and cells were re-suspended by the basal culture medium containing the conditioned culture medium. The cell suspension was inoculated to a well of a 12-well culture plate (which is coated with FNCcoatingmix in advance) and cultured under 5% CO.sub.2 at 37 C. The culture medium was replaced for the first time after 48 hours, and subsequently every other day. After the cell fusion reaches 100%, the cells are sub-cultured at a ratio of 1:2. The primary culture of Hcecs and the detection of markers thereof are shown in FIG. 2. The Hcecs cultured in vitro in the present disclosure are shown in the left picture of FIG. 6. It is observed that the cells are approximately hexagonal. The left picture of FIG. 6 shows living Hcecs which are hexagonal.

[0060] 3. Functions of Sub-Cultured Hcecs and Detection of Related Markers

[0061] Sub-culturing: Hcecs were continuously cultured by the basal culture medium containing the conditioned culture medium from orbital adipose-derived stem cells. The Hcecs were sub-cultured once every 3 to 5 days, at least over 13 generations, while maintaining the polygonal morphology and functions of the cells.

[0062] Detection of the proliferation capacity of Hcecs (Hcecs of the ninth, eleventh, thirteenth and fourteenth generations cultured in vitro) by scratch tests: as shown in FIG. 3, Hcecs of the ninth, eleventh and thirteenth generations can completely repair the scratch within 12 hours, and the Hcecs of the fourteenth generation cannot completely repair the scratch within 12 hours. The results show that Hcecs before the fourteenth generation have high proliferation capacity.

[0063] Detection of Related Markers:

[0064] As a tight junction protein between cells, N-cadherin is expressed in developing corneal endothelial cells.

[0065] As a tight junction protein between corneal endothelial cells, ZO-1 is distributed at tight junctions of normal corneal endothelial cells, and is an important constitute of the barrier function of corneal endothelium.

[0066] Na+/K+ATPase is presented in the cytoplasm and membrane of normal corneal endothelial cells and is a functional protein essential for the pump function of the corneal endothelial cells.

[0067] The N-Cadherin, ZO-1 and Na+/K+ATPase were detected by immunofluorescence. The experimental results are shown in FIG. 4.2. Hcecs sub-cultured over multiple generations express the N-Cadherin, ZO-1 and Na+/K+ATPase.

[0068] Detection by westernblotting was performed (the cells of the fifth, ninth, eleventh, ninth, eleventh and thirteenth generations, corresponding to P5, P9, P11, P9, P11 and P3 in FIG. 4.1). The experimental results are shown in FIG. 4.1. Hcecs sub-cultured over multiple generations express ZO-1 and Na+/K+ATPase.

[0069] 4. Verification of the Repair Capacity of Sub-Cultured Hcecs by Experiments on Animals

[0070] (1) Modeling of corneal endothelial function insufficiency of New Zealand white rabbits and rhesus monkeys: endothelium was removed by surgery.

[0071] (2) Hcec transplantation experiments: Hcecs of the eleventh generation cultured in vitro were injected into the anterior chamber; after the operation, examinations by slit lamps and by anterior segment optical coherence tomography (AS-OCT) were regularly performed as to the change in cornea; and after the operation, histological examination and other examinations were also performed on the cornea.

[0072] The results of experiments on animals are shown in FIG. 5 (FIG. 5.1 shows the result of experiments on the New Zealand white rabbits, FIG. 5-2 shows the result of experiments on the rhesus monkeys, upper and middle pictures are slit-lamp pictures, and the lower picture is the AS-OCT picture). Models of animals suffering from decompensation of corneal endothelium were established, and Hcecs were then injected into the anterior chamber and observed).

[0073] The results show that, as shown in FIG. 5.1, after the treatment of 7 days, by injecting Hcecs into the anterior chamber, the corneal edema and nubecula of the New Zealand white rabbits are gradually alleviated and the cornea finally becomes transparent, and the thickened cornea gradually becomes thinner and finally becomes basically normal.

[0074] As shown in FIG. 5.2, after the treatment of 7 days, by injecting Hcecs into the anterior chamber, the corneal edema and nubecula of the rhesus monkeys are gradually alleviated and the cornea finally becomes transparent, and the thickened cornea gradually becomes thinner and finally becomes basically normal.

[0075] Conclusion: the Hcecs cultured by the method of the present disclosure can recover the transparent cornea of animals suffering from decompensation of corneal endothelium. The cell therapy effect of Hcecs is fully proved.

[0076] The embodiments are merely preferred implementations of the present disclosure, and the implementations of the present disclosure are not limited thereto. Any other alternations, modifications, replacements, combinations and simplifications made without departing from the spirit essence and principle of the present disclosure shall be regarded as equivalent substitutions and shall fall into the protection scope of the present disclosure.