CN111979176B - Preparation method of human corneal epithelial cells, conditioned medium thereof and preparation method thereof - Google Patents

Preparation method of human corneal epithelial cells, conditioned medium thereof and preparation method thereof Download PDF

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CN111979176B
CN111979176B CN202010699887.3A CN202010699887A CN111979176B CN 111979176 B CN111979176 B CN 111979176B CN 202010699887 A CN202010699887 A CN 202010699887A CN 111979176 B CN111979176 B CN 111979176B
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刘佳
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Shenzhen Zhongjia Biomedical Technology Co ltd
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Abstract

The invention relates to a preparation method of human corneal epithelial cells, a conditioned medium and a preparation method thereof, and provides suitable seed cells, a suitable medium and an in vitro induction method to construct tissue engineering corneal epithelium. The preparation method of the human corneal epithelial cells comprises the following steps: primary culture, subculture and purification of hAMSCs; preparing a corneal epithelial cell conditioned medium; the conditioned medium is used for inducing hAMSCs to form corneal epithelial cells in vitro. Experiments prove that the hAMSCs can be differentiated into the human corneal epithelial cells by adopting the method, and preferably, the hAMSCs can be differentiated into the human corneal epithelial cells under the induction of a conditioned medium by adopting a BM + 55% -65% CM culture method.

Description

Preparation method of human corneal epithelial cells, conditioned medium thereof and preparation method thereof
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a preparation method of human corneal epithelial cells, a conditioned medium and a preparation method of the human corneal epithelial cells.
Background
Ocular trauma (mechanical injury, chemical injury, thermal burn, injury from surgical sources, etc.) or diseases (infection, cicatricial pemphigoid, etc.) can destroy the Limbal Stem Cell (LSCs) LSCs microenvironment, cause partial or complete Limbal Stem Cell Deficiency (LSCD), and further cause persistent corneal epithelial defects, corneal neovascularization, corneal metaplasia, scarring, etc., leading to vision loss, even corneal blindness.
The main current methods of treating LSCD are: 1. transplantation of cultured cells in vitro, such as oral mucosal epithelial cells. 2. Transplantation of non-cultured cells, such as autologous or allogeneic limbal transplantation. The above methods have certain effects in clinical treatment, but still face many difficulties, such as failure of providing stable cell phenotype by oral mucosal epithelial cells, limited source of LSCs donor, easy surgical injury caused by material selection, immunological rejection of allogeneic limbal transplantation, etc.
The development of tissue engineered corneal epithelium has created new promise for the treatment of LSCD. The seed cells expanded in vitro are planted on a carrier with good biocompatibility by utilizing a tissue engineering technology and cultured under an in vitro proper condition, so that tissues with morphological structures, physiological characteristics and gene expression similar to natural structures are obtained, a treatment strategy is provided for corneal epithelium reconstruction, and the method is a hotspot of current research in ophthalmology.
The seed cells are key elements for constructing the tissue engineering corneal epithelium and have certain capacities of amplification, differentiation and tissue formation, and the human LSCs are ideal cell sources, but the application of the LSCs is limited by the shortage of donors, so that the search for the proper seed cells is an urgent problem to be solved for constructing the tissue engineering corneal epithelium.
Disclosure of Invention
The invention aims to provide a preparation method of human corneal epithelial cells, a conditioned medium and a preparation method thereof, and provides suitable seed cells, a suitable medium and an in vitro induction method to construct tissue engineering corneal epithelium.
Therefore, the invention provides a preparation method of human corneal epithelial cells, which comprises the following steps: primary culture, subculture and purification of hAMSCs; preparing a corneal epithelial cell conditioned medium; the conditioned medium is used for inducing hAMSCs to form corneal epithelial cells in vitro.
In some embodiments, further comprising:
hAMSCs are differentiated into human corneal epithelial cells by a culture method of human serum-free mesenchymal stem cell culture medium BM and 30% -90% conditioned medium CM.
hAMSCs are differentiated into human corneal epithelial cells by a culture method of a human serum-free mesenchymal stem cell culture medium BM + 55% -65% CM conditioned medium.
The preparation of the corneal epithelial cell conditioned medium comprises the following steps: placing the treated rabbit corneal epithelium and the pre-elastic layer in a dish with the diameter being small, and enabling the corneal epithelium to face downwards; adding appropriate amount of 0.1-0.25% trypsin, digesting for 8-15 min at 35-40 deg.C, adding neutralization culture medium to stop digestion, centrifuging at 800-; cell resuspension was seeded in 35mm wellsPlacing into a dish, adding 4-6% CO at 35-40 deg.C2Culturing in a cell culture box, and changing the culture solution every 2 days; carrying out subculture when the cell growth density reaches 80-90%; recovering the 3 rd generation rabbit corneal epithelial cells, collecting cell culture fluid every 20-30 hours after the cells grow and fuse to 80% -90%, and obtaining a P4-P6 generation conditioned medium.
Inducing in vitro corneal epithelial cells of hAMSCs with conditioned medium comprises: mixing a conditioned medium CM and a human serum-free mesenchymal stem cell culture medium BM, and respectively preparing culture media with the CM proportion of 30-90%; washing 3 rd generation hAMSCs with good growth state with PBS, digesting with 0.1-0.25% trypsin for 1-5 min, adding human serum-free mesenchymal stem cell culture medium to terminate digestion, and centrifuging at 1200r/min for 1-10 min; discarding supernatant, adding appropriate amount of BM, 10%, 20%, 30%, 40%, 60%, 90%, and 100% of CM, respectively, and adjusting cell concentration to 0.8-1.2 × 105Per mL; inoculating cells into a 96-well plate, adding 80-100 mu L of cell suspension into each well, and arranging 2-5 multiple wells in each group; culturing in cell incubator for 20-30h, adding 5-15 μ LCCK-8 per well, and incubating in cell incubator at 35-40 deg.C under dark condition for 1-3 h.
Inducing in vitro corneal epithelial cells of hAMSCs with conditioned medium further comprises: washing primary hAMSCs with good growth state for one time; adding appropriate amount of 0.1-0.25% trypsin, digesting for 2-3min, and gently blowing with disposable pipette to completely separate adherent cells; (3) adding a human serum-free mesenchymal stem cell culture medium to terminate digestion, and centrifuging at 800-; (4) discarding the supernatant, and adding appropriate amount of BM, 30% CM, 60% CM, and 90% CM to obtain cell suspension; (5) the cells were inoculated in 24-well plates with a slide, named BM, 30% CM, 60% CM, 90% CM, respectively, and labeled on the surface of the plate, and the medium was changed every 2 days.
The primary culture, subculture and purification of hAMSCs comprise: adopting an improved homogenization method combined with an enzyme digestion and tissue block method to extract hAMSCs.
The invention also provides a conditioned medium, which is characterized by being prepared by the following method: placing the treated rabbit corneal epithelium and the pre-elastic layer in a dish with the diameter being small, and enabling the corneal epithelium to face downwards; adding proper amount of the mixtureDigesting for 8-15 minutes under the condition of 35-40 ℃ by 0.1-0.25 percent of trypsin, adding a neutralization culture medium to stop the digestion, centrifuging for 2-8 minutes at the speed of 800-; inoculating cell heavy suspension into 35mm dish, placing at 35-40 deg.C and 4-6% CO2Culturing in a cell culture box, and changing the culture solution every 2 days; carrying out subculture when the cell growth density reaches 80-90%; recovering the 3 rd generation rabbit corneal epithelial cells, collecting cell culture solution every 20-30 hours after the cells grow and fuse to 80% -90%, and obtaining the conditioned medium.
The invention also provides a preparation method of the conditioned medium, which is characterized by comprising the following steps: placing the treated rabbit corneal epithelium and the pre-elastic layer in a dish with the diameter being small, and enabling the corneal epithelium to face downwards; adding a proper amount of 0.1-0.25% trypsin for digesting for 8-15 minutes at 37 ℃, adding a neutralization culture medium to stop the digestion, centrifuging for 2-8 minutes at 800-; inoculating cell heavy suspension into 35mm dish, placing at 35-40 deg.C and 4-6% CO2Culturing in a cell culture box, and changing the culture solution every 2 days; carrying out subculture when the cell growth density reaches 80-90%; recovering the 3 rd generation rabbit corneal epithelial cells, collecting cell culture solution every 20-30 hours after the cells grow and fuse to 80% -90%, and obtaining the conditioned medium.
Experiments prove that the hAMSCs can be differentiated into the human corneal epithelial cells by adopting the method, and preferably, the hAMSCs can be differentiated into the human corneal epithelial cells under the induction of a conditioned medium by adopting a BM + 55% -65% CM culture method.
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FIG. 1 is a schematic representation of morphological observations under a microscope of primary and subcultures of hAMSCs according to one embodiment of the invention.
Fig. 2A and 2B are schematic diagrams of the identification and purity analysis of hAMSCs by detecting 3 rd generation hAMSCs phenotype molecules by flow cytometry according to an embodiment of the present invention, respectively, when CD44 and CD90 are added.
Fig. 3A and 3B are schematic diagrams respectively illustrating identification and purity analysis of hAMSCs by detecting 3 rd generation hAMSCs phenotype molecules using a flow cytometer according to an embodiment of the present invention, wherein C73 and CD105 are added.
FIG. 4 is a schematic diagram of the identification and purity analysis of hAMSCs by flow cytometry for the detection of 3 rd generation hAMSCs phenotype molecules according to an embodiment of the present invention when CD34+ CD19+ CD45+ CD11b + HLA-DR is added.
FIG. 5 is a graph showing the growth of hAMSCs by CCK-8 assay.
FIG. 6 shows an example of the CCK-8 method of the present invention for determining the effect of different concentrations of conditioned medium on the proliferation activity of hAMSCs.
FIG. 7 is a schematic representation of induced cell morphology according to one embodiment of the present invention.
FIG. 8 is a diagram showing the expression of CK3 in immunofluorescence assay according to an embodiment of the present invention, wherein A-D is a control group, E-H is a 30% CM group, I-L is a 60% CM group, and M-P is a 90% CM group.
FIG. 9 is a diagram showing the immunofluorescence assay for CK12 expression in one embodiment of the present invention, wherein A-D is a control group, E-H is a 30% CM group, I-L is a 60% CM group, and M-P is a 90% CM group.
FIG. 10 is a schematic diagram of a technical route of an embodiment of the present invention.
Detailed Description
The construction of tissue engineering corneal epithelium based on stem cells is the focus of research attention of the following embodiments of the invention, has wide sources, high proliferation capacity and no ethical problem, and can provide an ideal seed cell source for the construction of tissue engineering corneal epithelium.
The following embodiments of the present invention are based on Human amniotic mesenchymal stem cells (hAMSCs), and induce differentiation to Human corneal epithelial cells in vitro, thereby establishing a foundation for corneal epithelial tissue engineering. The tissue engineering corneal epithelium based on stem cells is constructed by taking the discarded placenta surface amnion from a lying-in woman, and the material has no additional damage to a human body, high cell proliferation speed and no ethical limit. Not only has strong proliferation capacity, but also has the characteristics of high purity, high abundance, high activity and the like. Has the characteristics of stem cells: low immunogenicity and multi-directional differentiation potential.
In the following embodiments of the present invention, innovations in stem cell induction method, preparation of Human amniotic mesenchymal stem cells (hmscs) and identification by flow cytometry, preparation of Conditioned Medium (CM) and Human serum-free mesenchymal stem cell medium (BM) mixed culture medium, and detection of expression of corneal epithelial cell markers CK3 and CK12 by immunofluorescence method are respectively involved.
The following embodiments of the invention adopt CM induction method for in vitro stem cell induction, the method is simple and easy to operate, and can make the inducing component fully contact with the cell and avoid the interference of other cells. The specific method comprises the following steps:
primary culture, subculture and purification of hAMSCs
Peeling placental fetal amniotic membrane under sterile condition, transferring to laboratory at 3-5 deg.C, and treating with penicillin-containing double antibody (penicillin 0.5-1.5 × 10)5U/L, streptomycin 0.5-1.5X 105U/L) is repeatedly washed for 1-5 times, the surface of amnion is repeatedly scraped by a cell scraper, tunica connective tissue is removed, and blood stain is washed and cleaned by the DPBS liquid.
Shearing placenta amnion with scissors into pieces of about 0.8-1.2cm, squeezing with forceps, repeatedly washing with physiological saline to clarify, squeezing blood and water with forceps, and weighing with electronic balance to obtain wet weight of amnion. Then adding a proper amount of normal saline, processing the mixture to be fine particles (about the size of rice grains) by using a handheld electric homogenizer, repeatedly washing the mixture by using the normal saline until the mixture is clear, and then continuously processing the mixture by using the homogenizer to be meat paste (with slight granular sensation and free suction by using a suction pipe); centrifuging at 1200-1800r/min for 2-8min, removing supernatant, adding pancreatin and II-type collagenase of 0.5-1.0g/L equal volume to the amniotic tissue into the precipitate, and digesting for 1.5-2.0h in a water bath at 35-40 ℃ with shaking.
After washing twice with physiological saline, the medium was added and inoculated in an amount of one T75 flask per 5-15g of amniotic tissue. Placing at 35-40 deg.C with volume fraction of 5% CO2And culturing in a saturated humidity incubator. Changing culture medium every 3d for 1 time, observing cell adherence by inverted phase contrast microscopeAnd the growth condition and the form change are photographed and recorded.
Observing the fusion rate of hAMSCs to about 75-85% by inverted phase contrast microscope, adding 0.1-0.25% pancreatin at 35-40 deg.C, and volume fraction of CO 4-6%2Digesting in an incubator for 2-5min to make adherent cells fall off and become round, then adding an equal volume of human serum-free mesenchymal stem cell culture medium to stop digestion, centrifuging at the rotating speed of 1200-1800r/min for 5-8min, removing supernatant, and adding 0.5-2mL of human serum-free mesenchymal stem cell culture medium to blow and resuspend cells. Mixing, sucking 30-60 μ L liquid with pipette into 0.2-1mL EP tube, sucking 5-15 μ L stem cell counting plate, and counting cells under microscope (cell number (/ m L) ═ 4 × 10 (total number of 4 large lattices)4Finally at 1-2X 108The concentration of the cells is between 65 and 85cm2In culture flasks, hAMSCs were transferred to 3 rd generation cell plates for testing.
Experiments prove that: the method for extracting the hAMSCs improves the advantages of a homogenization method combined with an enzyme digestion and tissue block method. The cell number is large, the cell can grow adherent to the wall in vitro, the growth speed is high, and the subculture is feasible. After 1-2 passages, a large amount of hAMSCs with higher purity can be obtained.
The method ensures that the digested hAMSCs are easier to attach to a culture vessel, the cell culture solution is easier to permeate, the opportunity that the cells contact the culture solution is increased, the cells can climb out easily, the tissue light transmittance is good, the observation under a microscope is convenient, and the extraction efficiency is high.
Taking 3 rd generation hAMSCs cells with fusion rate of 80% -90%, digesting and preparing the cells with density of 0.5-2 × 104one/mL cell suspension, 80-100. mu.L was added to a 96-well plate. Adding 80-100 μ LPBS solution into four side holes (total 36 holes) of 96-well plate, placing the cell culture plate at 35-40 deg.C, 4% -6% CO2After culturing for 20-30h, 8-12 μ L of CCK-8 solution is carefully added to 5 auxiliary wells, and the plate is incubated for 2-4h in the incubator every 20-25 h. The absorbance at 450nm was measured by the microplate reader and then repeated for 7 days. Data were recorded daily and proliferation curves were plotted.
Identification of hAMSCs phenotypes by flow cytometry
Melting3 rd generation with the integration rate of more than 80-90%, discarding culture solution, washing with PBS for 2 times, adding 0.1-0.25% pancreatin 0.01-0.5% EDTA digestive solution, digesting in 37 deg.C incubator for 2-5min, adding equal volume stop solution to stop digestion, blowing and mixing the digested cells, centrifuging at the speed of 1200-1800r/min for 2-8min, discarding supernatant, blowing and resuspending cells with PBS, centrifuging at the speed of 1200-1800r/min for 2-8min, repeating for 1-5 times, adjusting cell concentration to 1-3 × 109L-1, putting into flow tubes, adding 80-100 mu L of cell suspension into each tube, adding mouse anti-human monoclonal antibodies CD44-PE, CD90-FITC, CD105-Per CP-Cy, CD73-APC and negative control mixed solution CD34-PE, CD19-PE, CD45-PE, CD11b-PE and HLA-DR at room temperature in a dark place, incubating for 20-40min in a dark place at room temperature, adding 1-3m LPBS into each flow tube, fully shaking, centrifuging for 2-8min at 1800r/min, discarding supernatant, adding 200-300 mu LPBS for resuspension, identifying by a flow cytometer, and calculating the cell surface antigen positive rate.
Preparation of conditioned Medium for corneal epithelial cells
(1) After a New Zealand white rabbit is killed by a 1-5% sodium pentobarbital solution, an eyeball is quickly removed and the new Zealand white rabbit is placed in PBS containing a 1% streptomycin solution;
(2) repeatedly rinsing eyeball with PBS containing 1% streptomycin solution, and placing in sterile culture dish;
(3) the puncture knife punctures the eyeball along the corneosclera margin and takes the corneal tissue by the ophthalmology scissors;
(4) repeatedly washing with PBS containing 1% streptomycin solution to remove excessive tissue on the inner surface of cornea;
(5) tearing off the elastic layer, and repeatedly washing with PBS containing 1% streptomycin solution;
(6) placing the corneal epithelium and the pre-elastic layer in a dish with a diameter of 35mm with the corneal epithelium facing down;
(7) adding appropriate amount of 0.1-0.25% trypsin, digesting for 8-15 min at 35-40 deg.C, adding neutralization culture medium to stop digestion, centrifuging at 800-;
(8) the cell suspension was inoculated into a 35mm dish and placed35-40℃、4-6％CO2Culturing in a cell culture box, and changing the culture solution every 2 days;
(9) and (5) carrying out subculture when the cell growth density reaches 80-90%.
(10) Recovering the 3 rd generation rabbit corneal epithelial cells, collecting cell culture fluid every 20-30 hours after the cells grow and fuse to 80% -90%, and obtaining Conditioned Medium (CM).
CCK-8 method for detecting influence of different proportions of conditioned medium on cell proliferation activity
(1) Mixing CM (conditioned medium) and BM (human serum-free mesenchymal stem cell culture medium) to respectively prepare culture media with CM proportion of 10%, 20%, 30%, 40%, 60%, 90% and 100%;
(2) washing 3 rd generation hAMSCs with good growth state with PBS, digesting with 0.1-0.25% trypsin for 1-5 min, adding human serum-free mesenchymal stem cell culture medium to terminate digestion, and centrifuging at 1200r/min for 1-10 min;
(3) discarding supernatant, adding appropriate amount of BM, 10%, 20%, 30%, 40%, 60%, 90%, and 100% of CM, respectively, and adjusting cell concentration to 0.8-1.2 × 105Per mL;
(4) inoculating cells into 96-well plate, adding 80-100 μ L cell suspension into each well, and setting 2-5 multiple wells in each group
(5) Culturing in a cell incubator for 20-30h, adding 5-15 mu LCCK-8 into each hole, and incubating in the cell incubator at 37 ℃ under the condition of keeping out of the sun for 1-3 h;
(6) and detecting the OD value of 460nm wavelength by using a microplate reader.
Observation form of different culture medium proportions under microscope for in vitro induction of hAMSCs corneal epithelial cells
(1) Washing primary hAMSCs with good growth state for one time;
(2) adding appropriate amount of 0.1-0.25% trypsin, digesting for 2-3min, and gently blowing with disposable pipette to completely separate adherent cells;
(3) adding a human serum-free mesenchymal stem cell culture medium to terminate digestion, and centrifuging at 800-;
(4) discarding the supernatant, and adding appropriate amount of BM, 30% CM, 60% CM, and 90% CM to obtain cell suspension;
(5) inoculating the cells into 24-well plate containing reptile, respectively named BM, 30% CM, 60%
CM, 90% CM, marking on the surface of the culture plate, and changing the culture solution every 2 days;
(6) cell immunofluorescence experiments were performed on days 3, 7, 11, and 14 after induction culture, and expression of corneal epithelial cell markers CK3 and CK12 was observed.
Immunofluorescence method for detecting expression of CK3 and CK12 in corneal epithelial cells
(1) Discarding the cell culture solution, washing with PBS for 2-5 times, each time for 2-5 min;
(2) adding appropriate amount of 4% paraformaldehyde to fix cells for 20-40min, discarding solution, washing with PBS for 2-5 times, each time for 2-8 min;
(3) treating with 0.1-0.5% TritonX-100 for 5-15min, discarding solution, washing with PBS for 3 times, each time for 10 min;
(4) blocking with 1-10% BSA for 20-40min, discarding the solution, washing with PBS for 2-5 times, each time for 2-8 min;
(5) respectively dripping mouse anti-human CK3(1:100) antibody and rabbit anti-human CK12(1:100) antibody, and incubating overnight at 2-8 ℃;
(6) discarding the primary antibody solution, washing with PBS for 2-5 times, each time for 5-15 min;
(7) adding a DyLight 488-labeled goat anti-mouse IgG (1:500) secondary antibody and a DyLight 594-labeled goat anti-rabbit IgG (1:500) secondary antibody dropwise, and incubating for 1h at room temperature in a dark condition;
(8) discarding the secondary antibody diluent, washing with PBS for 2-5 times, each time for 2-8 min;
(9) dropping DAPI staining solution to treat cells for 2-8min, discarding the staining solution, washing with PBS for 3 times, each time for 2-8 min;
(10) marking the glass slide, and dripping a trace of anti-fluorescence quenching agent on the glass slide;
(11) and taking out the slide, slightly absorbing the surface moisture by using absorbent paper, slightly placing the cell surface downwards on a glass slide on which an anti-fluorescence quencher is dripped for mounting, and observing and taking a picture under a fluorescence microscope.
The following examples demonstrate the feasibility of the invention experimentally due to the unclear cytokine and pathway mechanisms associated with cell differentiation. This experiment uses CM to simulate the corneal epithelial cell growth microenvironment to induce hAMSCs. The CM and the BM are mixed to prepare three culture media with different proportions to induce hAMSCs to differentiate into human corneal epithelial cells. In the experiment, Conditioned Medium (CM) and human serum-free mesenchymal stem cell medium (BM) are mixed to prepare culture medium with the proportion of 10%, 20%, 30%, 40%, 60%, 90% and 100% CM, which are respectively used for culturing hAMSCs and detecting cell proliferation activity by a CCK-8 method. hAMSCs are induced in vitro by 30%, 60% and 90% CM, hAMSCs cultured by BM are used as a control group, and the expression of corneal epithelial cell markers CK3 and CK12 is detected by an immunofluorescence method. At 3 days of induction, 30%, 60%, 90% of CM group cells did not express CK3, 60% and 90% of CM group cells began to express CK 12; at 7 days, the expression of CK3 and CK12 can be seen in 30%, 60% and 90% CM group cells; at 11 and 14 days, 30%, 60%, 90% CM group cells continued to express CK3, CK 12. At 3, 7, 11 and 14 days of induction, the expression of CK3 and CK12 is not seen in BM group cells.
Experiments prove that hAMSCs can be differentiated into human corneal epithelial cells by adopting a BM + 30% -90% CM culture method, more preferably, a BM + 55% -65% CM culture method is adopted, and the following examples illustrate the culture method with the optimal value of 60% CM; it was confirmed that hAMSCs were able to differentiate into human corneal epithelial cells under the induction of conditioned medium.
Example 1 Primary culture, subculture and purification of hAMSCs
Peeling placental fetal amniotic membrane under sterile condition, transferring to laboratory at 4 deg.C, and treating with penicillin 1 × 105U/L, streptomycin 1X 105U/L) sterile DPBS solution is repeatedly washed for 2 times, the surface of amnion is repeatedly scraped by a cell scraper, tunica connective tissue is removed, and blood stain is washed and cleaned by the DPBS solution.
Shearing placenta amnion with scissors into pieces of about 1cm, squeezing with forceps, repeatedly washing with physiological saline until clear, squeezing blood and water with forceps, and weighing with electronic balance to obtain wet weight of amnion. Then adding a proper amount of normal saline, processing the mixture to be fine particles (about the size of rice grains) by using a handheld electric homogenizer, repeatedly washing the mixture by using the normal saline until the mixture is clear, and then continuously processing the mixture by using the homogenizer to be meat paste (with slight granular sensation and free suction by using a suction pipe); centrifuging at 1500r/min for 5min, removing supernatant, adding pancreatin and type II collagenase in the same volume as amnion tissue into the precipitate, and digesting in 37 deg.C water bath tank under shaking for 1.5-2.0 h.
After washing twice with physiological saline, the medium was added and inoculated in an amount of one T75 flask per 10g of amniotic tissue. Placing at 37 ℃ with 5% CO by volume fraction2And culturing in a saturated humidity incubator. The culture medium is replaced every 3d for 1 time, and the adherent and growth conditions and morphological changes of the cells are observed by an inverted phase contrast microscope and photographed and recorded.
The fusion rate of hAMSCs reaches about 80% by inverted phase contrast microscope observation, 0.125% pancreatin is added at 37 ℃, and the volume fraction of CO is 5%2Digesting in an incubator for 2.5min to make adherent cells fall off and become round, then adding an equal volume of human serum-free mesenchymal stem cell culture medium to terminate digestion, centrifuging at a rotating speed of 1500r/min for 6min, removing supernatant, adding 1m L, and blowing and beating the resuspended cells by the human serum-free mesenchymal stem cell culture medium. After the mixture was blown and mixed, 40. mu.L of the liquid was aspirated into an EP tube of 0.5m L using a pipette, and 10. mu.L of a stem cell counting plate was aspirated to count the cells under a microscope, where the cell count (/ m L) ═ total of 4 cells/4). times.104Finally at 1.5X 108The cell concentration is 75cm2In culture flasks, hAMSCs were transferred to 3 rd generation cell plates for testing.
The morphological observation results of the hAMSCs under the microscope are shown in FIG. 1, and the results show that: the method for extracting the hAMSCs has the advantages of combining enzyme digestion and tissue block method by improving a homogenization method. The number of cells is large, the cells can grow in an adherent manner quickly in vitro, the growth speed is high, a small part of cells can be observed to grow in an adherent manner after being inoculated for 24 hours, and most of cells need to grow in an adherent manner for about 24-48 hours. The primary hAMSCs are observed to be in an oval, polygonal or irregular shape by an inverted phase contrast microscope and grow adherently. And 4-5 days later, the cell fusion rate reaches 80-90%, and the subculture can be performed. After the trypsination is added, the bulge shrinkage of hAMSCs can be observed under a mirror, the edge is spherical, the hAMSCs are easier to fall off from a flat bottom than cells such as amniotic epithelial cells and the like, and the adherence speed is higher than that of other miscellaneous cells. After 1-2 passages, a large amount of hAMSCs with high purity can be obtained, cells cultured by passage culture have the adherent time of about 2-4h, the shape is uniform, the cell volume is gradually increased along with the increase of the passage times, the proportion of cell nucleus is also gradually increased, most of the cells are in long fusiform shape, and part of the cells can be seen to grow in a whirlpool shape and a fish school shape (figure 1A, B, C, D, the magnification is 40 times)).
The method ensures that the digested hAMSCs are easier to attach to a culture vessel, the cell culture solution is easier to permeate, the opportunity that the cells contact the culture solution is increased, the cells can climb out easily, the tissue light transmittance is good, the observation under a microscope is convenient, and the extraction efficiency is high.
According to the existing data, foreign research on human fetal corneal cell culture has mostly focused on the mid-eighties of the last century. The corneal epithelial cells are separated from human abortion fetus, and can be obtained by primary or passage through trypsin digestive fluid. Whether transplanted with donated corneas or in vitro cultured corneal epithelial cells, donors are required to provide corneal tissue and are also limited by donor shortages, donor age, immune rejection, and the like. Furthermore, corneal epithelial cells directly isolated from human cornea, although having a certain proliferation ability in vitro, are difficult to culture continuously.
Stem cells are an ideal source of corneal epithelial cells due to their pluripotent and self-renewing properties. At present, bone marrow-derived mesenchymal stem cells are most extensive, and compared with bone marrow-derived placental amnion, hAMSCs have the advantages of sufficient source, low immunogenicity, low virus pollution rate, no social ethical dispute and the like. Bone marrow is derived from different donors, and individual differences in donor age, physical function, disease, etc. all affect the characteristics of MSCs, whereas hAMSCs completely avoid the above problems.
At present, the hMSCs separation culture method mainly comprises a tissue block method and a trypsin digestion method, the whole process is complicated to operate and easy to pollute, and the cell activity and the passage capacity are influenced due to too long enzyme treatment time.
In the method, (1) the amnion tissue is cut into small pieces only by hand, thereby reducing the labor consumption and time cost, and the amnion of a complete placenta can be completely processed within one working day. (2) Treating to fine granule (about rice grain size) with hand-held electric homogenizer, repeatedly washing with normal saline to remove most fetal blood, and further treating with homogenizer to meat paste (with slight granular feeling and capable of being freely sucked with suction tube); centrifuging at 500r/min for 5min, and removing supernatant. By this process, fetal blood and red blood cells therein can be substantially removed. (3) After the treatment of the homogenizer, the tissue blocks become very thin and tiny, the dispersion degree of the tissue blocks is increased, and the inoculation area is increased. (4) After being treated by the homogenizer, the mixture is organized into a meat paste shape, thereby reducing the digestion time of trypsin and avoiding the damage to cells. (5) The sediment before inoculation is pasty, and the tissue blocks are adhered to each other, so that the micro tissue is not easy to float after being added with the culture medium, and the micro tissue can have a better wall-adhering effect.
EXAMPLE 2CCK-8 assay for hAMSCs proliferation Activity
The 3 rd generation hAMSCs cells with the fusion rate of 85% are taken, digested and made into the density of 1 × 104one/mL of the cell suspension was added to a 96-well plate at 100. mu.L. The cells were incubated at 37 ℃ in a 96-well plate with 100. mu.L of LPBS solution in four wells (36 wells in total) and 5% CO2After 24 hours of incubation in the incubator of (1), 10. mu.L of a-CCK-8 solution was carefully added to 5 of the wells, and the plate was incubated in the incubator for 3 hours every 24 hours. The absorbance at 450nm was measured by the microplate reader and then repeated for 7 days. Data were recorded daily and proliferation curves were plotted.
The results are shown in FIG. 5. The CCK-8 detection result shows that: the subcultured hAMSCs grow slowly at 1d and have weak proliferation capacity, but the proliferation activity is increased to different degrees along with the increase of time, the logarithmic growth phase is reached at 4d, the plateau phase is reached at 5-6d, and the proliferation rate is basically unchanged. Proliferation Activity and proliferation Curve (FIG. 5)
Example 3 identification of hAMSCs phenotypes by flow cytometry
Collecting 3 rd generation with fusion rate above 85%, discarding culture solution, washing with PBS for 2 times, addingAdding pancreatin with concentration of 0.125% -EDTA digestive juice with concentration of 0.02%, digesting in 37 deg.C incubator for 3min, adding equal volume stop solution to stop digestion, blowing and mixing the digested cells uniformly, centrifuging at 1500r/min for 6min, discarding supernatant, blowing and blowing heavy suspension cells with PBS, centrifuging at 1500r/min for 6min, repeating for 2 times, and adjusting cell concentration to 2 × 109And L-1, filling the mixture into flow tubes, adding 100 mu L of cell suspension into each tube, adding mouse anti-human monoclonal antibodies CD44-PE, CD90-FITC, CD105-Per CP-Cy, CD73-APC and negative control mixed liquor CD34-PE, CD19-PE, CD45-PE, CD11b-PE and HLA-DR at room temperature in a dark place, incubating the mixture for 30min at room temperature in a dark place, adding 2m LPBS into each flow tube, fully shaking the mixture, centrifuging the mixture for 6min at 1500r/min, discarding supernatant, adding 250 mu LPBS to resuspend cells, identifying the cells by a flow cytometer, and calculating the positive rate of cell surface antigens.
The identification results are shown in FIG. 2A and FIG. 4: flow cytometry detection of phenotype 3 rd generation hAMSCs results of flow cytometry detection show that: high expression of CD73, CD90, CD105 and CD44, low expression of CD34, CD19, CD45, CD11b and HLA-DR. (FIGS. 2 to 4)
EXAMPLE 4 corneal epithelial cell conditioned Medium preparation
(1) After a New Zealand white rabbit is killed by a 3% sodium pentobarbital solution, an eyeball is quickly removed and the new Zealand white rabbit is placed in PBS containing a 1% streptomycin solution;
(2) repeatedly rinsing eyeball with PBS containing 1% streptomycin solution, and placing in sterile culture dish;
(3) the puncture knife punctures the eyeball along the corneosclera margin and takes the corneal tissue by the ophthalmology scissors;
(4) repeatedly washing with PBS containing 1% streptomycin solution to remove excessive tissue on the inner surface of cornea;
(5) tearing off the elastic layer, and repeatedly washing with PBS containing 1% streptomycin solution;
(6) placing the corneal epithelium and the pre-elastic layer in a dish with a diameter of 35mm with the corneal epithelium facing down;
(7) adding appropriate amount of 0.125% trypsin, digesting at 37 deg.C for 10min, adding neutralization culture medium to stop digestion, centrifuging at 1000r/min for 5min, and resuspending in human serum-free mesenchymal stem cell culture medium;
(8) the cell suspension was inoculated into a 35mm dish and placed at 37 ℃ in 5% CO2Culturing in a cell culture box, and changing the culture solution every 2 days;
(9) and (5) carrying out subculture when the cell growth density reaches 80-90%.
(10) Recovering the 3 rd generation rabbit corneal epithelial cells, collecting cell culture fluid every 24 hours after the cells grow and fuse to 80% -90%, and obtaining Conditioned Medium (CM).
The conventional methods for culturing corneal epithelial cells in vitro include a serum culture method and a serum-free culture method, wherein the serum-free culture method includes a tissue block method and a digestive enzyme method. Digestive enzymes are gradually eliminated, and tissue block methods are increasingly used for in vitro corneal epithelial cell culture. Different laboratories can adopt different methods to culture corneal epithelial cells in vitro, and the efficiency and the purity are greatly different.
The method is improved on the traditional technology, and the cornucopia is completely cornucopia paster of a culture dish, so that the operation is simple and easy, the experimental operation time is reduced, and the possibility of cell mixing can be well reduced.
Example 4CCK-8 method to examine the Effect of different ratios of conditioned Medium on cell proliferation Activity
(1) Mixing CM (conditioned medium) and BM (human serum-free mesenchymal stem cell culture medium) to respectively prepare culture media with CM proportion of 10%, 20%, 30%, 40%, 60%, 90% and 100%;
(2) taking 3 rd generation hAMSCs with good growth state, washing with PBS, digesting with 0.125% trypsin for 2 min, adding human serum-free mesenchymal stem cell culture medium to terminate digestion, and centrifuging at 1000r/min for 5 min;
(3) discarding the supernatant, adding appropriate amount of BM, 10%, 20%, 30%, 40%, 60%, 90%, and 100% of CM, respectively, and adjusting cell concentration to 1 × 105Per mL;
(4) cells were seeded in 96-well plates, 100. mu.L of cell suspension was added per well, 3 multiple wells per group
(5) Culturing in a cell incubator for 24h, adding 10 mu LCCK-8 into each hole, and incubating in the cell incubator for 2h at 37 ℃ in a dark condition;
(6) and detecting the OD value of 460nm wavelength by using a microplate reader.
The detection results are shown in fig. 6: using BM, 10%, 20%, 30%, 40%, 60%, 90%, 100% of CM to culture hAMSCs, using CCK-8 method to detect the effect of different culture media on cell proliferation activity, comparing the cell proliferation activity of different groups, the result shows: no significant difference in cell proliferation activity (FIG. 6)
The long-term culture and clinical application of corneal epithelial cells in vitro face many challenges. (1) Most of the culture medium formulas for long-term culture need to be added with bovine serum, and the risk of pathogen contamination and the instability of culture conditions exist. (2) The proliferation capacity of corneal epithelial cells is limited, and the cells are easy to change in morphology after long-term culture and tend to reduce the morphology of fibroblasts and the expression of functional proteins. (3) Most of the cultured human corneal epithelial cells are from old donors, have short passage time and poor proliferation capacity, and are more prone to irregular cells.
(4) Cell lines established by gene transfection techniques present the risk of neoplasia and virus cytotoxicity problems. How to widen the source of the human corneal epithelial cells and optimize long-term culture is realized, so that the corneal epithelial cells can still well maintain the cell morphology, the functional phenotype and the proliferation capacity after passage, and potential infection, tumor formation and cytotoxicity are avoided, thus becoming a research hotspot.
In order to make the human corneal epithelium cultured in vitro closer to corneal epithelial cells in a normal physiological state, the present inventors have searched for a corneal epithelial cell culture medium. The media currently used in most corneal epithelial cell studies are DMEM and DMEM/F12. Basic medium supplements commonly used include bFGF, NGF, EGF, vitamin C, B27, calcium chloride, and the like. In primary culture of corneal epithelial cells, it is difficult to promote cell adhesion and proliferation at a serum concentration of less than 20% without adding other nutrients and pro-cell growth factors. Serum has many disadvantages.
The improved culture scheme has the advantages that (1) the basic culture medium is a human serum-free mesenchymal stem cell culture medium, fetal bovine serum is not required to be added, the conditioned medium is culture supernatant obtained after the human mesenchymal serum-free culture medium is used for culturing rabbit corneal epithelial cells for P3 generations, particularly P4-P6 generations, and the conditions of risk of heterologous protein or pathogen pollution, unclear batch components and the like do not exist. (2) The hAMSCs-derived human corneal epithelial cells have characteristics of both siccative and corneal epithelial cells of hAMSCs stem cells, can be stably proliferated, do not change cell morphology after culture, and tend to reduce fibroblast morphology, functional protein expression and other problems. (3) The cultured human corneal epithelial cells come from the placenta of a newborn fetus, the passage time is long, the proliferation capacity is strong, and the cell morphology is uniform. (4) The corneal epithelial cell line established without gene transfection technology has the risk of tumor formation and the cytotoxicity problem of virus.
We collected the conditioned medium which is the culture supernatant of corneal epithelial cells after culturing the P4-P6 generation human serum-free mesenchymal stem cells. Theoretically, the stem cells are dry more strongly the earlier the cell generation. However, in the process of in vitro culture of stem cells, the stem cells have a regulation and adaptation process from a tissue microenvironment to an in vitro culture environment, and a part of cells which are not adapted can be eliminated, so that in the first two generations, the stem cell genome has unstable factors, and is probably not suitable for clinical application. The research shows that the stem cells have abnormal karyotype in the first 3 generations of cells in a high proportion through karyotype analysis. For example, Mesenchymal Stem Cells (MSCs), the optimal generation for clinical use should be between P4 and P6.
Therefore, in order to broaden the sources of human corneal epithelial cells and improve in vitro culture conditions, the human placental amniotic mesenchymal stem cells are tried to be induced and differentiated into the human corneal epithelial cells under different serum-free culture medium combination conditions, namely CM (conditioned medium) and BM (human serum-free mesenchymal stem cell culture medium) are matched in different proportions, so that the complexity and uncertainty of the culture conditions caused by the addition of animal serum can be avoided, and the normal proliferation and differentiation of the corneal cells can be ensured.
Example 5 in vitro induction of hAMSCs into corneal epithelial cells with different Medium ratios morphology under microscope
(1) Washing primary hAMSCs with good growth state for one time;
(2) adding appropriate amount of 0.125% trypsin, digesting for 2-3min, and gently blowing with disposable pipette to completely separate adherent cells;
(3) adding a human serum-free mesenchymal stem cell culture medium to terminate digestion, and centrifuging at 1000r/min for 5 min;
(4) discarding the supernatant, and adding appropriate amount of BM, 30% CM, 60% CM, and 90% CM to obtain cell suspension;
(5) inoculating cells into a 24-pore plate with a slide, respectively naming BM, 30% CM, 60% CM and 90% CM, marking the surface of the culture plate, and changing the culture solution every 2 days;
(6) cell immunofluorescence experiments were performed on days 3, 7, 11, and 14 after induction culture, and expression of corneal epithelial cell markers CK3 and CK12 was observed.
The observations are shown in FIGS. 7A-O: induction cell morphology observation induction was 1 day and 3 days, and cell morphology did not change significantly (fig. 7A, 7B, 7F, 7G, 7K, 7L), and was typically long spindle; on day 7, the cell volume began to increase, with irregular wide flat, polygonal, elliptical, etc. (FIGS. 7C, 7H, 7M); on day 11 and 14, the cells of different CM groups presented increasing proportions with irregular changes in morphology (FIGS. 7D, 7E, 7I, 7J, 7N, 7O)
Example 6 immunofluorescence assay for detecting the expression of CK3 and CK12 in corneal epithelial cells
(1) Discarding the cell culture solution, washing with PBS for 3 times, 5min each time;
(2) adding appropriate amount of 4% paraformaldehyde to fix cells for 30min, discarding solution, washing with PBS for 3 times, 5min each time;
(3) treating with 0.2% TritonX-100 for 10min, discarding the solution, washing with PBS for 3 times, each time for 10 min;
(4) blocking with 5% BSA for 30min, discarding the solution, washing with PBS for 3 times, 5min each time;
(5) respectively dripping mouse anti-human CK3(1:100) antibody and rabbit anti-human CK12(1:100) antibody, and incubating overnight at 4 ℃;
(6) discarding the primary antibody solution, washing with PBS for 3 times, each time for 10 min;
(7) adding a DyLight 488-labeled goat anti-mouse IgG (1:500) secondary antibody and a DyLight 594-labeled goat anti-rabbit IgG (1:500) secondary antibody dropwise, and incubating for 1h at room temperature in a dark condition;
(8) discarding the secondary antibody diluent, washing with PBS for 3 times, 5min each time;
(9) dropping DAPI staining solution to treat cells for 5min, discarding the staining solution, washing with PBS for 3 times, 5min each time;
(10) marking the glass slide, and dripping a trace of anti-fluorescence quenching agent on the glass slide;
(11) and taking out the slide, slightly absorbing the surface moisture by using absorbent paper, slightly placing the cell surface downwards on a glass slide on which an anti-fluorescence quencher is dripped for mounting, and observing and taking a picture under a fluorescence microscope.
The detection results are shown in FIGS. 8A-N and 9A-O, respectively: at 3 days of induction, 30%, 60%, 90% of CM group cells did not express CK3 (fig. 8E, 8I, 8M), and 60% and 90% of CM group cells began to express CK12 (fig. 9I, 9M); at 7 days, CK3 and CK12 were expressed in 30%, 60% and 90% CM cells (FIGS. 8F, 8J and 8N; FIGS. 9F, 9J and 9N); at 11 and 14 days, 30%, 60%, 90% of the CM groups continued to express CK3, CK12 (FIG. 8G, 8H, 8K, 8L, 8O, 8P; FIG. 9G, 9H, 9K, 9L, 9O). The expression of CK3 and CK12 was not observed in BM group cells (FIGS. 8A-8D; FIGS. 9A-9D).
Experiments prove that hAMSCs can be differentiated into human corneal epithelial cells by adopting a BM + 30% -90% CM culture method; it was confirmed that hAMSCs were able to differentiate into human corneal epithelial cells under the induction of conditioned medium.

Claims (7)

1. A preparation method of human corneal epithelial cells is characterized by comprising the following steps:
primary culture, subculture and purification of hAMSCs; preparing a corneal epithelial cell conditioned medium;
inducing hAMSCs into corneal epithelial cells in vitro by using a conditioned medium;
the preparation of the corneal epithelial cell conditioned medium comprises the following steps: placing the treated rabbit corneal epithelium and the pre-elastic layer in a small dish to enable the corneal epithelium to face downwards; adding appropriate amount of 0.1-0.25% trypsin, digesting for 8-15 min at 35-40 deg.C, adding neutralization culture medium to stop digestion, centrifuging at 800-; inoculating the cell heavy suspension into a 35mm dish, culturing in a 4-6% CO2 cell culture box at 35-40 deg.C, and changing the solution every 2 days; carrying out subculture when the cell growth density reaches 80-90%; recovering rabbit corneal epithelial cells of the 3 rd generation, collecting cell culture solution every 20-30 hours after the cells grow and fuse to 80% -90%, and obtaining a P4-P6 generation conditioned medium;
hAMSCs are differentiated into human corneal epithelial cells by a culture method of human serum-free mesenchymal stem cell culture medium BM and 30% -90% conditioned medium CM.
2. The method of claim 1, wherein the hAMSCs are differentiated into human corneal epithelial cells by culturing in a medium conditioned with human serum-free mesenchymal stem cell medium BM plus 55% -65% CM.
3. The method of claim 1, wherein inducing hAMSCs into corneal epithelial cells in vitro using conditioned medium comprises: mixing a conditioned medium CM and a human serum-free mesenchymal stem cell culture medium BM, and respectively preparing culture media with the CM proportion of 30-90%; washing 3 rd generation hAMSCs with good growth state with PBS, digesting with 0.1-0.25% trypsin for 1-5 min, adding human serum-free mesenchymal stem cell culture medium to terminate digestion, and centrifuging at 1200r/min for 1-10 min; discarding supernatant, adding appropriate amount of BM, 30%, 40%, 60%, and 90% of CM, respectively, and adjusting cell concentration to 0.8-1.2 × 105Per mL; inoculating cells into a 96-well plate, adding 80-100 mu L of cell suspension into each well, and arranging 2-5 multiple wells in each group; culturing in cell incubator for 20-30h, adding 5-15 μ LCCK-8 per well, and incubating in cell incubator at 35-40 deg.C under dark condition for 1-3 h.
4. The method of claim 3, wherein inducing hAMSCs into corneal epithelial cells in vitro using conditioned medium further comprises: washing primary hAMSCs with good growth state for one time; adding appropriate amount of 0.1-0.25% trypsin, digesting for 2-3min, and gently blowing with disposable pipette to completely separate adherent cells; (3) adding a human serum-free mesenchymal stem cell culture medium to terminate digestion, and centrifuging at 800-; (4) discarding the supernatant, and adding appropriate amount of BM, 30% CM, 60% CM, and 90% CM to obtain cell suspension; (5) the cells were inoculated in 24-well plates with a slide, named BM, 30% CM, 60% CM, 90% CM, respectively, and labeled on the surface of the plate, and the medium was changed every 2 days.
5. The method of claim 1, wherein the hAMSCs primary culture, subculture and purification comprises: adopting an improved homogenization method combined with an enzyme digestion and tissue block method to extract hAMSCs.
6. A conditioned medium, characterized in that it is prepared by the following method: placing the treated rabbit corneal epithelium and the pre-elastic layer in a small dish to enable the corneal epithelium to face downwards; adding appropriate amount of 0.1-0.25% trypsin, digesting for 8-15 min at 35-40 deg.C, adding neutralization culture medium to stop digestion, centrifuging at 800-; inoculating cell heavy suspension into 35mm dish, placing at 35-40 deg.C and 4-6% CO2Culturing in a cell culture box, and changing the culture solution every 2 days; carrying out subculture when the cell growth density reaches 80-90%; recovering the 3 rd generation rabbit corneal epithelial cells, collecting cell culture fluid every 20-30 hours after the cells grow and fuse to 80% -90%, and obtaining a P4-P6 generation conditioned medium.
7. A method of preparing a conditioned medium, comprising: placing the treated rabbit corneal epithelium and the pre-elastic layer in a small dish to enable the corneal epithelium to face downwards; adding appropriate amount of 0.1-0.25% trypsin, digesting for 8-15 min at 35-40 deg.C, adding neutralization culture medium to stop digestion, centrifuging at 800-; inoculating cell heavy suspension into 35mm dish, placing at 35-40 deg.C and 4-6% CO2Culturing in cell culture box, and separating 2Changing the liquid every day; carrying out subculture when the cell growth density reaches 80-90%; recovering the 3 rd generation rabbit corneal epithelial cells, collecting cell culture fluid every 20-30 hours after the cells grow and fuse to 80% -90%, and obtaining a P4-P6 generation conditioned medium.
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