CN110079500B - Limbal stem cells and methods of making the same - Google Patents
Limbal stem cells and methods of making the same Download PDFInfo
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Abstract
The invention relates to a high-activity limbal stem cell and a preparation method thereof, wherein the preparation method comprises the following steps: obtaining corneal tissue, and pretreating the corneal tissue to be used; adding a cell cryopreservation solution containing a cell inducer into the pretreated corneal tissue; freezing the corneal tissue with the cell freezing solution; the cell inducer is an agent that induces inflammation or stress in cells in corneal tissue locally, but does not directly cause cell death. The method can realize the screening of the limbal stem cells and various mature and differentiated corneal cells in corneal tissues by simple low-temperature freezing operation before cell culture, and can realize the long-term freezing storage of the high-activity limbal stem cells.
Description
Technical Field
The invention relates to the field of tissue engineering, in particular to a preparation method of limbal stem cells.
Background
The limbus is the transitional zone between the cornea and the sclera, and since the clear cornea is embedded in the opaque sclera and gradually transitions into the sclera, there is no clear demarcation on the surface and histology of the eyeball. The limbus is a natural storage adult stem cell position which is responsible for corneal epithelium regeneration and repair, the limbus stem cells are arranged in a palisade way at the base of an epithelial layer in the limbus tissue and are a high-quality cell source for ocular surface corneal epithelium ulcer and tissue engineering lamellar corneal reconstruction, and the limbus stem cell transplantation vegetation is considered to be one of the most promising treatment methods aiming at the ocular surface diseases at present.
Limbal stem cells play an important role in corneal ocular surface reconstruction, but since limbal stem cells are easily differentiated during in vitro culture and are difficult to expand in large quantities, it is necessary to obtain primary limbal stem cells by a more gentle method and to find a better method for maintaining the stem cell activity of the cells in vitro. In order to obtain limbal stem cells from corneal tissue, the current methods for obtaining limbal stem cells mainly include: enzyme digestion and tissue mass adherence. The enzyme digestion method mainly comprises treating and digesting corneal tissue with trypsin, collagenase or neutral protease at 33 deg.C, digesting corneal epithelial layer, and inoculating to culture dish for culture; the tissue block adherence method comprises the steps of shearing a corneal limbus tissue block with a certain size, adhering the corneal limbus tissue block to the bottom of a culture dish, and adding a culture solution to allow the corneal limbus tissue block to migrate out of cells for culture. The enzyme digestion method can obtain more cells, but the purity of the limbal stem cells is lower; the tissue block adherence method obtains purer cells, but the tissue block adherence time and the cell migration time are longer.
Although the above methods can isolate limbal stem cells, there are still problems of low activity and easy differentiation of limbal stem cells in the subsequent in vitro culture. In order to maintain the dryness of limbal stem cells, cells are often expanded in vitro using methods such as cell co-culture, vector culture, and media supplemented with growth factors. The cell co-culture method is to directly culture the limbal stem cells on feeder cells, and growth factors and cytokines secreted by the feeder cells can keep the limbal stem cells dry, but the use of the feeder cells easily causes cell cross contamination, and the difficulty of subsequent cell separation is high. Amniotic membrane is a good carrier for culturing the limbal stem cells, and many studies show that the limbal stem cells cultured on the amniotic membrane have good dryness, high survival rate and rapid growth, but the limbal stem cells separated from the amniotic membrane also have the problem of cell cross contamination. The addition of cell growth factors to the culture medium can also promote the growth of limbal stem cells, and for example, epidermal growth factor, hepatocyte growth factor, basic fibroblast growth factor, keratinocyte growth factor and the like can promote mitosis, cell migration and stem cell proliferation, but at present, the type and proportion of the added growth factors are not defined.
Although various methods are used for maintaining the activity of the limbal stem cells in culture, some cells are inevitably differentiated, and in order to obtain better effects in practical applications such as limbal stem cell transplantation, tissue engineering lamellar cornea construction, and corneal disease mechanism research, the limbal stem cells need to be screened to obtain high-activity stem cells, and the current screening methods include a flow sorting method, an immunomagnetic bead method, a collagen adsorption sorting method and the like. The flow sorting method is a method for screening marked cells by a flow cytometer, the immunomagnetic bead screening is a method for sorting cells by retaining the cells through a magnetic field with certain strength by utilizing the specific binding of magnetic beads coated with antibodies and special marks on the surfaces of stem cells according to the immunological principle, and the flow sorting method and the immunomagnetic bead method are common cell screening methods. The collagen adsorption sorting method is a method for screening the limbal stem cells with high stem cell activity by the long adsorption time of the cells on the extracellular matrixes such as collagen and the like according to the rapid adsorption capacity of the epithelial stem cells on the extracellular matrixes, and the method is high in sorting speed and avoids the influence on the cell activity due to long time or the toxicity of special substances, but the method is a relatively rough screening method and cannot accurately screen the limbal stem cells.
The limbal stem cells that have not been used for a while are often preserved, and the preservation method of limbal stem cells that is widely used at present is a low-temperature cryopreservation method, which is a general procedureThe following were used: after digestion and dispersion of cells, cooling with cryoprotectant (10% glycerol or 3.5% -10% dimethyl sulfoxide in culture medium containing 10% serum), and diluting cell suspension to (2-5) × 106And/ml, subpackaging the cell suspension according to the amount of 1 ml/ampoule, placing the sealed ampoule into a slow freezer or a programmed cooling instrument, slowly freezing at the speed of reducing the temperature by 1 ℃ per minute, slowly freezing the cell suspension added with the protective agent at the temperature of-25 ℃, and placing the frozen ampoule into a low-temperature freezer or a liquid nitrogen tank. The existing cell preservation solution has simple nutrient components, no fixed proportion and single preservation method, and causes low cell survival rate and poor cell stability of stem cells, and the activity of the stem cells cannot be well maintained. The cornea tissue which is not used in a short period is usually placed in cornea preservation liquid and preserved at the temperature of 2-2 ℃, the preservation time is 2 weeks, when the preservation time exceeds 2 weeks, a method adopted by the cornea tissue preservation liquid comprises an organ culture preservation method and a deep low temperature preservation method, both methods need special preservation liquid and preservation equipment, and the cornea is rarely preserved for a long period in practical application because the number of apoptosis cells of the cornea is continuously increased and the structure is gradually disordered along with the increase of the preservation time.
The common characteristics of the above methods for culturing, screening and storing limbal stem cells are that the culturing, screening and storing of limbal stem cells are performed after the cells are obtained from fresh corneal tissue, and the conventional cell screening steps are required, the operation process is complicated, each step may cause the loss of limbal stem cells, and it is difficult to successfully obtain a large number of high-activity limbal stem cells.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a method for producing limbal stem cells, which can realize the screening of limbal stem cells and mature and differentiated various types of corneal cells in corneal tissues by adding a cell inducer to corneal tissues and a simple low-temperature freezing operation before cell culture, and can realize the long-term cryopreservation of highly active limbal stem cells, which does not require any special equipment, is easy and convenient to operate, has high screening efficiency and high screening cell purity, and can greatly improve the proportion and activity of limbal stem cells.
The specific technical scheme for realizing the technical purpose is as follows:
a method for preparing high-activity limbal stem cells, comprising the steps of:
obtaining corneal tissue, and pretreating the corneal tissue to be used;
adding a cell cryopreservation solution containing a cell inducer into the pretreated corneal tissue;
freezing the corneal tissue with the cell freezing solution;
the cell inducer is an agent that induces inflammation or stress in cells in corneal tissue locally, but does not directly cause cell death.
In some of these embodiments, the cell inducing agent is selected from one or more of an inflammatory factor, a hormone, a modulator of an intracellular signaling molecule, and a modulator of the cell cycle.
In some of these embodiments, the inflammatory factor is selected from one or more of an interleukin, an interferon, and a tumor necrosis factor; the hormone is selected from one or more of glucocorticoid, mineralocorticoid and sex hormone; the intracellular signaling molecule modulator is selected from one or more of actinomycetes, protein kinase C activators, protein kinase C inhibitors and DNA topoisomerase inhibitors; the cell cycle modulator is selected from one or more of cyclin-dependent kinase inhibitors, cyclins, cyclin-dependent kinase inhibitors, G1-specific cyclins, G2/mitosis-specific cyclins, and transcription or degradation factors thereof.
In some of these embodiments, the cell inducing agent is selected from one or more of a glucocorticoid, a protein kinase C inhibitor, and a tumor necrosis factor.
In some of these embodiments, the cell inducing agent is selected from one or more of prednisolone acetate, staurosporine, and tumor necrosis factor α.
In some of these embodiments, the cell cryopreservation solution is a mixture of a cell culture solution containing the cell inducer and a cryoprotectant; the volume ratio of the cryoprotectant to the cell culture solution containing the cell inducer is 1: 2-20.
In some of these embodiments, the volume ratio of the cryoprotectant to the cell culture fluid containing the cell inducer is 1: 2-10.
In some of these embodiments, the cryoprotectant is selected from one or more of dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide, methanol, polyvinylpyrrolidone, fructose, sucrose, dextran, albumin, polyethylene glycol, ethyl starch, and egg yolk.
In some of these embodiments, the cell inducer is present in the cell culture fluid at a concentration of 1ng/ml to 100. mu.g/ml.
In some of these embodiments, the cell inducer is present in the cell culture fluid at a concentration of 1ng/ml to 10. mu.g/ml.
In some of these embodiments, the cell inducing agent is present in the cell culture fluid at a concentration of 10ng/ml to 5. mu.g/ml.
In some of these embodiments, the cell inducer is present in the cell culture fluid at a concentration of 4-4.5. mu.g/ml.
In some of these embodiments, the cell culture fluid is DMEM high-glucose supplemented with: 0-50 wt% of fetal bovine serum, 0-20 wt% of glutamine, 0-20 wt% of nonessential amino acids, 0-20 wt% of penicillin-streptomycin solution, 0-1000ng/ml of epidermal growth factor, 0-1000 mu g/ml of transferrin, 0-1000 mu g/ml of insulin and 1ng/ml-10 mu g/ml of the cell inducer.
In some of these embodiments, the cell culture fluid is DMEM high-glucose supplemented with: 2-12 wt% of fetal bovine serum, 0.2-1.2 wt% of glutamine, 0.2-1.2 wt% of nonessential amino acids, 0.2-1.2 wt% of penicillin-streptomycin solution, 2-12ng/ml of epidermal growth factor, 4-6 mu g/ml of transferrin, 4-6 mu g/ml of insulin and 10ng/ml-5 mu g/ml of the cell inducer.
In some of these embodiments, the cell culture fluid is DMEM high-glucose supplemented with: 10 wt% fetal calf serum, 1 wt% glutamine, 1 wt% nonessential amino acids, 1 wt% penicillin-streptomycin solution, 10ng/ml epidermal growth factor, 5 mug/ml transferrin, 5 mug/ml insulin, 4-4.5 mug/ml of the cell inducer.
In some of these embodiments, the freezing temperature is between 0 ℃ and-200 ℃.
In some of these embodiments, the freezing temperature is between-20 ℃ and-196 ℃.
In some of these embodiments, the freezing time is 1-120 months.
In some of these embodiments, the freezing time is 1-24 months.
In some of these embodiments, the freezing time is 3-6 months.
In some of these embodiments, the method of preparing limbal stem cells further comprises the steps of: and (5) separating and culturing the frozen corneal tissue to obtain the limbal stem cells.
In some of these embodiments, the cell culture medium used in the culturing is supplemented with 1ng/ml to 100. mu.g/ml of the cell inducer.
In some of these embodiments, the cell culture medium used in the culturing is supplemented with 1ng/ml to 10. mu.g/ml of the cell inducer.
In some of these embodiments, the cell culture medium used in the culturing is supplemented with 1ng/ml to 5. mu.g/ml of the cell inducer.
In some of these embodiments, the cell culture medium used in the culturing is supplemented with 4-4.5. mu.g/ml of the cell inducer.
In some of these embodiments, the cell culture fluid used for the culture is high-glucose DMEM supplemented with: 0-50 wt% of fetal bovine serum, 0-20 wt% of glutamine, 0-20 wt% of nonessential amino acids, 0-20 wt% of penicillin-streptomycin solution, 0-1000ng/ml of epidermal growth factor, 0-1000 mu g/ml of transferrin, 0-1000 mu g/ml of insulin and 1ng/ml-10 mu g/ml of the cell inducer.
In some of these embodiments, the cell culture fluid is DMEM high-glucose supplemented with: 2-12 wt% of fetal bovine serum, 0.2-1.2 wt% of glutamine, 0.2-1.2 wt% of nonessential amino acids, 0.2-1.2 wt% of penicillin-streptomycin solution, 2-12ng/ml of epidermal growth factor, 4-6 mu g/ml of transferrin, 4-6 mu g/ml of insulin and 10ng/ml-5 mu g/ml of the cell inducer.
In some of these embodiments, the cell culture fluid used for the culture is high-glucose DMEM supplemented with: 10 wt% fetal calf serum, 1 wt% glutamine, 1 wt% nonessential amino acids, 1 wt% penicillin-streptomycin solution, 10ng/ml epidermal growth factor, 5 mug/ml transferrin, 5 mug/ml insulin, 4-4.5 mug/ml of the cell inducer.
In some of these embodiments, the method of isolation culture is selected from one or more of tissue adherence and digestion.
In some of these embodiments, the tissue walling method comprises the steps of: after the limbal tissue is separated from the corneal tissue, the limbal tissue epithelial layer is attached to a culture vessel and cultured, and the limbal stem cells are migrated from the limbal tissue to obtain limbal stem cells. Preferably, the isolated corneal limbal tissue is between 0.1mm and 20mm in size and between 0.01mm and 3mm thick.
In some of these embodiments, the digestion process comprises the steps of: the corneal epithelial layer containing the limbal stem cells is digested and separated from corneal tissue using a digestive enzyme, and then the corneal epithelial layer is inoculated into a culture vessel and cultured to obtain the limbal stem cells. Preferably, the digestive enzyme is any one or more of collagenase, neutral protease and trypsin.
In some of these embodiments, the pre-processing comprises: the corneal tissue to be used is routinely washed, disinfected and isolated using physiological buffers with or without antibiotics.
In some of these embodiments, the corneal tissue is selected from any one or more of human, rodent, and other mammalian corneal tissues.
In some of these embodiments, the corneal tissue is any one of fresh corneal tissue or corneal tissue that has undergone short-term storage, wherein the short-term storage is from 1 day to 20 days.
Another object of the present invention is to provide a highly active limbal stem cell.
The specific technical scheme is as follows:
the limbal stem cells produced by the above method for producing limbal stem cells.
The method for preparing the limbal stem cells comprises the steps of adding a cryopreservation solution containing a cell inducer into corneal tissues, and then freezing at low temperature to screen and store the cells, wherein the added cell inducer can induce cells in the corneal tissues to locally generate inflammation or apoptosis, and the low-temperature freezing condition can induce adult cells in the corneal tissues to generate apoptosis or cell necrosis and simultaneously maintain and store the activity of the limbal stem cells in the corneal tissues, so that the purposes of screening and long-term storage of the limbal stem cells can be achieved, and the method can obtain the high-activity limbal stem cells; and then the frozen corneal tissue is isolated and cultured, and the limbal stem cells with high proportion can be further obtained.
The principle and beneficial effects of the preparation method of the high-activity limbal stem cells of the invention are as follows:
(1) effectively realize the screening of the limbal stem cells and various mature and differentiated corneal cells in corneal tissues: under the action of cell inducer, corneal tissue can generate inflammatory stress by matching with low-temperature freezing condition treatment. Since endothelial cells, stromal cells and epithelial cells in corneal tissue are mature differentiated cells and do not have the ability to resist this inflammatory stress, apoptosis or necrosis gradually occurs under cryo-freezing conditions. Because the limbal stem cells have the characteristics of large nuclear-to-cytoplasmic ratio, slow renewal cycle and the like, the limbal stem cells in the tissues can resist the stimulation and do not undergo apoptosis, and are maintained and preserved. Therefore, the cornea tissue can be removed of other mature differentiated cells and the limbal stem cells can be retained through simple freezing treatment, and the screening efficiency and the screening cell purity are high.
(2) With the help of natural stem cell niche advantages, limbal stem cells and their stem cell activity are maintained and preserved: the palisade tissue in the limbus is the storage niche for limbal stem cells. The limbal stem cells are protected from inflammation or stress conditions by the presence of beneficial factors such as abundant extracellular matrix, growth factors, specific three-dimensional stromal architecture, etc. within the limbal natural niche. Therefore, with these advantages, the limbal stem cells in the corneal tissue can maintain their stem cell activity under cryo-freezing conditions, while achieving long-term storage of the limbal stem cells.
(3) In the screening process, the in vivo inflammatory stress state is simulated, so that the activation of the limbal stem cells in the culture process can be accelerated: limbal stem cells are responsible for the renewal and repair of the corneal epithelial layer in vivo. These signals are transmitted to limbal locations as mature differentiated cells in the corneal epithelium, stroma undergo progressive apoptosis or necrosis. Following receipt of the damage signal by the limbal stem cells, repair will begin under appropriate conditions. Thus, when corneal tissue is removed from cryo-frozen conditions for primary cell culture, limbal stem cells can be rapidly activated and proliferate and migrate from the tissue.
(4) The method of the invention has simple operation and low production cost: compared with screening methods such as a flow type sorting method, an immunomagnetic bead method, a collagen adsorption sorting method and the like, the method disclosed by the invention can be used for screening the cells in the corneal tissue only by adding a cell inducer and performing low-temperature freezing treatment, does not need specific instruments and equipment, and greatly reduces the cost in the aspects of reagent consumables, instrument use, human resources and the like.
(5) The method can improve the proportion and the activity of the limbal stem cells to a greater extent, and the limbal stem cells with higher activity can be obtained by the method, can be used for basic research such as the construction of cell models in the mechanism research of keratopathy, can also provide a high-quality seed cell source for the field of tissue engineering, or can promote the development of the limbal stem cell transplantation for treating eye surface diseases.
Drawings
FIG. 1 is a graph showing the results of Caspase 1(Caspase 1) staining of fresh cornea and cornea treated with cell inducer and cryogenically treated in example 1;
FIG. 2 is a graph showing the results of flow cytometry of fresh cornea and primary cultured limbal stem cells from treated corneal tissue in example 1;
FIG. 3 is a graph showing immunofluorescence staining and western blot of member 2 of the ATP-binding transporter G superfamily (ABCG2) in example 1;
FIG. 4 is a graph showing the results of detecting the relative expression amounts of tumor necrosis factor alpha, interleukin 6 and interleukin 1 beta of cornea by RT-qPCR in example 2;
FIG. 5 is a graph showing the result of staining K3, P63 in a corneal tissue section obtained in example 2;
FIG. 6 is a graph of flow cytometry assay results for fresh versus treated corneal tissue primary cultured limbal stem cells in example 2.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In one embodiment of the present invention, a method for preparing limbal stem cells is provided, comprising the steps of:
obtaining corneal tissue, and pretreating the corneal tissue to be used;
adding a cell cryopreservation solution containing a cell inducer into the pretreated corneal tissue;
freezing the corneal tissue with the cell freezing solution;
the cell inducer is an agent that induces inflammation or stress in cells in corneal tissue locally, but does not directly cause cell death.
The method can achieve the purposes of screening and long-term preservation of the limbal stem cells by adding the cryopreservation liquid containing the cell inducer into the corneal tissue and then freezing at low temperature to screen and preserve the cells, wherein the added cell inducer can induce local inflammation or apoptosis of the cells in the corneal tissue, and the low-temperature freezing condition can induce apoptosis or necrosis of adult cells in the corneal tissue, and simultaneously maintain and preserve the activity of the limbal stem cells in the corneal tissue, so that the method can obtain the high-activity limbal stem cells.
In some of these embodiments, the cell inducing agent is preferably one or more of an inflammatory factor, a hormone, an intracellular signaling molecule modulator, and a cell cycle modulator that induces inflammation or stress locally to cells in the corneal tissue, but does not directly cause cell death.
Among the possible inflammatory factors, include, but are not limited to, interleukins, interferons, tumor necrosis factors, transforming growth factors, chemokines, and the like; possible hormones include, but are not limited to, corticosteroids, sex hormones, growth hormones, thyroxine, insulin, adrenal hormones, and the like; possible intracellular signaling molecule modulators include, but are not limited to, actinomycetes, protein kinase C activators, protein kinase C inhibitors, DNA topoisomerase inhibitors, cytoplasmic tyrosine kinase inhibitors, phosphatidylinositol kinase inhibitors, apoptosis signal-regulating kinase inhibitors, and the like; possible cell cycle modulators include, but are not limited to, cyclin-dependent kinase inhibitors, cyclins, cyclin-dependent kinase inhibitors, G1-specific cyclins, G2/mitosis-specific cyclins and transcription or degradation factors thereof, histone deacetylase inhibitors, Janus kinase inhibitors, and the like.
Further preferably, the inflammatory factor is selected from one or more of interleukins, interferons and tumor necrosis factors; the hormone is selected from one or more of glucocorticoid, mineralocorticoid and sex hormone; the intracellular signaling molecule modulator is selected from one or more of actinomycetes, protein kinase C activators, protein kinase C inhibitors and DNA topoisomerase inhibitors; the cell cycle modulator is selected from one or more of cyclin-dependent kinase inhibitors, cyclins, cyclin-dependent kinase inhibitors, G1-specific cyclins, G2/mitosis-specific cyclins, and transcription or degradation factors thereof.
Further preferably, the cell inducing agent is selected from one or more of a glucocorticoid (which may be, for example, prednisolone acetate), a protein kinase C inhibitor (which may be, for example, staurosporine), and a tumor necrosis factor (which may be, for example, tumor necrosis factor alpha (TNF-alpha)).
In some embodiments, the cell inducer is effective by adding to the cell cryopreservation solution, which can be an existing conventional cryopreservation solution, preferably a mixture of a cell culture solution containing the cell inducer and a cryoprotectant; the preferred volume ratio of the cryoprotectant to the cell culture fluid containing the cell inducer is 1: 2-20, more preferably in a volume ratio of 1: 2-10. Wherein the cryoprotectant is selected from one or more of dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide, methanol, polyvinylpyrrolidone, fructose, sucrose, dextran, albumin, polyethylene glycol, ethyl starch and egg yolk; dimethyl sulfoxide is preferred.
The cell culture solution in the frozen stock solution may be a conventional culture solution for culturing limbal stem cells. Preferably DMEM high-glucose added with the cell inducer and the following components: 0-50 wt% fetal bovine serum, 0-20 wt% glutamine, 0-20 wt% nonessential amino acids, 0-20 wt% penicillin-streptomycin solution, 0-1000ng/ml Epidermal Growth Factor (EGF), 40-1000 μ g/ml transferrin, 0-1000 μ g/ml insulin. More preferably DMEM of high glucose type to which the cell inducer and the following components are added: 2-12 wt% of fetal bovine serum, 0.2-1.2 wt% of glutamine, 0.2-1.2 wt% of non-essential amino acid, 0.2-1.2 wt% of penicillin-streptomycin solution, 2-12ng/ml of epidermal growth factor, 4-6 mu g/ml of transferrin and 4-6 mu g/ml of insulin. Most preferably DMEM high glucose added with the cell inducer and the following components: 10 wt% fetal bovine serum, 1 wt% glutamine, 1 wt% nonessential amino acids, 1 wt% penicillin-streptomycin solution, 10ng/ml epidermal growth factor, 5. mu.g/ml transferrin, 5. mu.g/ml insulin. The addition amount of the cell inducer is preferably 1ng/ml-100 mug/ml, and when the addition amount of the cell inducer is within the range, the inflammation or stress reaction locally generated by cells in the corneal tissue can be moderate, so that mature differentiated cells such as endothelial cells, stromal cells and epithelial cells in the corneal tissue can be subjected to apoptosis or necrosis to the maximum extent under the subsequent low-temperature freezing condition, meanwhile, the limbal stem cells can be resisted by the stimulation of the cell inducer to the maximum extent without apoptosis, and the proportion and the activity of the limbal stem cells can be maintained and preserved to the maximum extent. More preferably in an amount of 10ng/ml to 10. mu.g/ml, still more preferably in an amount of 10ng/ml to 5. mu.g/ml, still more preferably in an amount of 4 to 4.5. mu.g/ml.
In some of these embodiments, the temperature of the freezing is from 0 ℃ to-200 ℃. Preferably-20 ℃ to-196 ℃, under which the apoptosis or the necrosis of the adult cells in the corneal tissue can be better induced, and the activity of the limbal stem cells in the corneal tissue can be better maintained and preserved, so that the proportion and the activity of the obtained limbal stem cells can be further improved.
In some of these embodiments, the freezing time is from 1 to 120 months. In this time frame, it is possible to sufficiently induce apoptosis or necrosis of adult cells in corneal tissue, and to preserve the activity of limbal stem cells to a high degree. The period of time is further preferably 1 to 24 months, more preferably 3 to 6 months.
Further, the method for producing limbal stem cells of the present invention may further comprise the step of culturing the frozen corneal tissue separately. The corneal tissue after freezing is separated and cultured by a conventional method, and the limbal stem cells with high proportion and high activity can be further obtained.
The cell culture solution for culturing the limbal stem cells in the method of the present invention may be a conventional culture solution, and is preferably high-glucose DMEM supplemented with the cell inducer and the following components: 0-50 wt% of fetal calf serum, 0-20 wt% of glutamine, 0-20 wt% of nonessential amino acid, 0-20 wt% of penicillin-streptomycin solution, 0-1000ng/ml of epidermal growth factor, 0-1000 mu g/ml of transferrin and 0-1000 mu g/ml of insulin. More preferably DMEM of high glucose type to which the cell inducer and the following components are added: 2-12 wt% of fetal bovine serum, 0.2-1.2 wt% of glutamine, 0.2-1.2 wt% of non-essential amino acid, 0.2-1.2 wt% of penicillin-streptomycin solution, 2-12ng/ml of epidermal growth factor, 4-6 mu g/ml of transferrin and 4-6 mu g/ml of insulin. Most preferably DMEM high glucose added with the cell inducer and the following components: 10 wt% fetal bovine serum, 1 wt% glutamine, 1 wt% nonessential amino acids, 1 wt% penicillin-streptomycin solution, 10ng/ml epidermal growth factor, 5. mu.g/ml transferrin, 5. mu.g/ml insulin. Wherein the cell inducer is preferably added in an amount of 1ng/ml to 100. mu.g/ml, more preferably in an amount of 10ng/ml to 10. mu.g/ml, more preferably in an amount of 10ng/ml to 5. mu.g/ml, more preferably in an amount of 4 to 4.5. mu.g/ml.
In the method provided by the invention, the isolated culture method can be a conventional cell culture method, and can be one or more of a tissue adherence method and a digestion method. The tissue adherence method comprises the following steps: after the limbal tissue is separated from the corneal tissue, the limbal tissue epithelial layer is attached to a culture vessel and cultured, and the limbal stem cells are migrated from the limbal tissue to obtain limbal stem cells. Preferably, the isolated corneal limbal tissue is between 0.1mm and 20mm in size and between 0.01mm and 3mm thick. The digestion method comprises the following steps: the corneal epithelial layer containing the limbal stem cells is digested and separated from corneal tissue using a digestive enzyme, and then the corneal epithelial layer is inoculated into a culture vessel and cultured to obtain the limbal stem cells. Preferably, the digestive enzyme is any one or more of collagenase, neutral protease and trypsin.
In some of these embodiments, the pre-treatment comprises: the corneal tissue to be used is routinely washed, disinfected and isolated using physiological buffers with or without antibiotics.
The corneal tissue used in the methods of the invention can be any one or more of human, rodent, and other mammalian corneal tissue. The corneal tissue is preferably any one of fresh corneal tissue or corneal tissue subjected to short-term preservation for 1 to 20 days. The limbal stem cells in the corneal tissue that satisfy this condition have higher activity, which is beneficial for subsequent screening, storage and culture.
The invention also provides the high-activity limbal stem cells prepared by the preparation method of the limbal stem cells, which can be used for constructing cell models in basic research such as mechanism research of keratopathy, can also provide a high-quality seed cell source for the field of tissue engineering, or can promote the development of the limbal stem cell transplantation for treating eye surface diseases.
The present invention will be described in further detail with reference to specific examples.
The percentages not specifically described in the following examples are percentages by mass.
Example 1: preparation of Rabbit limbal Stem cells
(1) Obtaining corneal tissue, pre-treating corneal tissue to be used:
after an eyeball taken down from a New Zealand rabbit is washed for 3 times by Phosphate Buffered Saline (PBS) added with 1% penicillin-streptomycin solution, redundant tissues around the outer part of the eyeball are removed by a tissue scissors, 1-5 mm sclera is reserved along the corneal limbus, the rest part is cut off, the content is removed, parts such as the iris are scraped clean, and then the part is washed for 3 times by the PBS.
(2) Adding a cell freezing medium containing a cell inducer to the pretreated corneal tissue:
firstly, preparing a cell culture solution KCM for culturing the limbal stem cells in vitro: high-glucose DMEM + 10% fetal bovine serum + 1% glutamine + 1% nonessential amino acids + 1% penicillin-streptomycin solution +10ng/ml Epidermal Growth Factor (EGF) + 5. mu.g/ml transferrin + 5. mu.g/ml insulin, and 4. mu.g/ml prednisolone acetate and 20ng/ml staurosporine (STS) are added, wherein the prednisolone acetate is glucocorticoid, the staurosporine is protein kinase C inhibitor, the two jointly act as a cell inducer, and then cell freezing stock solution is prepared: 10% DMSO plus 90% KCM medium (volume percent). Placing the corneal tissue pretreated in the step (1) into a 1.5ml cryopreservation tube, and adding 1ml of prepared cell cryopreservation liquid.
(3) The corneal tissue added with the cell freezing solution is frozen at low temperature to screen and store the corneal limbal stem cells:
and (3) putting the freezing tube in the step (2) into a program cooling box, placing the freezing tube into a refrigerator at the temperature of-20 ℃, and transferring the freezing tube into a liquid nitrogen tank after freezing.
(4) Separating and culturing the corneal tissue frozen in the step (3) to obtain the limbal stem cells:
six months later, the frozen corneal tissue was removed from the liquid nitrogen tank, rapidly thawed in a 33 ℃ water bath for 1 minute, and washed 3 times with PBS. The whole cornea (containing a small amount of sclera) epithelium was spread upward in a clean large dish, and a tissue mass (to include a transparent portion and an opaque portion) was drilled with a 3mm trephine with the limbus as a center line to obtain a limbal tissue mass. The limbal tissue blocks are digested in 0.1% IV collagenase for 15 minutes and then taken out, epithelial layers are dipped with a small amount of fetal calf serum and then are attached to a six-hole plate downwards, then the obtained mixture is placed into an incubator to be dried for 30 minutes, 1.5ml of KCM culture solution is added into each hole respectively for standing culture, cells are migrated out after 2-4 days, and the high-activity limbal stem cells subjected to cryopreservation screening can be obtained.
The cornea (treated group cornea) and fresh cornea after the addition of the cell inducer and the low-temperature freezing treatment were subjected to paraffin embedding, sectioning and immunofluorescence staining, respectively, and the results are shown in fig. 1. The results show that the epithelial structures of the two groups of corneal tissues are completely preserved, the stroma layers are not closely arranged, and the endothelial layers are exfoliated. In fresh corneas, Caspase 1 expression was not seen at the central cornea and limbal locations. In the treatment group, Caspase 1 was expressed in the central cornea throughout the layer, while a small amount of Caspase 1 expression was observed in the limbal surface layer and Caspase 1 expression was not observed in the basal position (white arrows in FIG. 1 indicate the expression positions). In corneal tissue, the central corneal site is the mature differentiated corneal epithelial cells and the limbal base is the limbal stem cells. When the cells are in a stress state, the cells express Caspase 1 and cause cell inflammation and apoptosis. Therefore, the above test results indicate that after the treatment with the cell-inducing agent and the low-temperature freezing treatment, the mature and differentiated corneal epithelial cells in the treated corneal tissue undergo inflammatory apoptosis, while the limbal stem cells in the tissue are not affected.
Flow cytometry was performed on the cornea (treated group cornea) after the addition of the cell inducer and the low-temperature freezing treatment and the primary cultured limbal stem cells of the fresh cornea, respectively, and the proportion of P63 positive cells was observed, and the results are shown in fig. 2. The results show that the proportion of cells P63 in the treated group was 95%, which is much higher than 32% of the untreated fresh corneal group. P63 is one of the limbal stem cell markers, the expression of which is closely related to the proliferative activity of stem cells. The detection result shows that the corneal tissue subjected to the cell inducer and the low-temperature freezing treatment can obtain more limbal stem cells with better proliferation activity.
Immunofluorescence staining and western blot detection are carried out on the cornea (treated group cornea) after being added with the cell inducer and being frozen at low temperature and the fresh cornea primary culture limbal stem cell, and the expression condition of the stem cell ABCG2 (adenosine triphosphate binding transport protein G superfamily member 2) is observed, and the result is shown in figure 3. The results show that the number of cells positively expressed by the ABCG2 in the treated group is far more than that in the fresh cornea group, and the results of the western blot semi-quantitative detection show that the expression amount of the ABCG2 in the cells in the treated group is higher than that in the fresh cornea group. ABCG2 is one of the markers of the limbal stem cells, and the detection result shows that the corneal tissue which is subjected to the cell inducer and the low-temperature freezing treatment can obtain more limbal stem cells with better activity.
The results show that the limbal stem cells obtained by the method for screening, storing and culturing the limbal stem cells have high proportion and can keep good stem cell activity.
Example 2: preparation of human limbal Stem cells
(1) Obtaining corneal tissue, pre-treating corneal tissue to be used:
cleaning human eyes with the clonidine eye drops for 5-6 times, then respectively washing with III-type aniodide and physiological saline added with gentamicin, taking down the eyeballs, removing redundant tissues around the outer parts of the eyeballs by using a tissue scissors, keeping 1-5 mm of sclera along the corneal limbus, cutting the rest parts, simultaneously removing contents, scraping off parts such as iris and the like, and then cleaning the cornea for 3 times by using Phosphate Buffer Solution (PBS) added with 1% penicillin-streptomycin solution.
(2) Adding a cell freezing medium containing a cell inducer to the pretreated corneal tissue:
firstly, preparing a cell culture solution KCM for culturing the limbal stem cells in vitro: high-glucose DMEM + 10% fetal bovine serum + 1% glutamine + 1% nonessential amino acid + 1% penicillin-streptomycin solution +10ng/ml Epidermal Growth Factor (EGF) +5 mug/ml transferrin +5 mug/ml insulin, and 4 mug/ml prednisolone acetate and 10ng/ml tumor necrosis factor alpha (TNF-alpha) are added, wherein the prednisolone acetate is glucocorticoid, the tumor necrosis factor alpha is inflammatory factor, the two act together as cell inducer, and then cell freezing solution is prepared: 10% dimethyl sulfoxide plus 90% KCM medium. Placing the corneal tissue pretreated in the step (1) into a 1.5ml cryopreservation tube, and adding 1ml of prepared cell cryopreservation liquid.
(3) The corneal tissue added with the cell freezing solution is frozen at low temperature to screen and store the corneal limbal stem cells:
and (3) putting the freezing tube in the step (2) into a programmed cooling box, placing the freezing box in a refrigerator at the temperature of-20 ℃, and transferring the freezing tube into a liquid nitrogen tank after overnight freezing.
(4) Separating and culturing the corneal tissue frozen in the step (3) to obtain the limbal stem cells:
three months later, the cryopreserved corneal tissue was removed from the liquid nitrogen tank, rapidly thawed in a 33 ℃ water bath for 1 minute, and washed 3 times with PBS. The whole corneal (containing a small amount of sclera) epithelium was spread upward in a clean dish and a 5mm size piece of limbal tissue was cut with scissors. The limbal tissue blocks are digested in 0.1% IV collagenase for 15 minutes and then taken out, epithelial layers are dipped with a small amount of fetal calf serum and then are attached to a six-hole plate downwards, then the obtained mixture is placed into an incubator to be dried for 30 minutes, 1.5ml of KCM culture solution is added into each hole respectively for standing culture, cells are migrated out after 2-4 days, and the high-activity limbal stem cells subjected to cryopreservation screening can be obtained.
The relative expression amounts of tumor necrosis factor alpha, interleukin 6 and interleukin 1 beta of the cornea were detected by RT-qPCR method, and the results are shown in fig. 4: compared with fresh cornea, after the cell inducer is added and the low-temperature freezing treatment is carried out, the transcription levels of three inflammatory factors of tumor necrosis factor alpha, interleukin 6 and interleukin 1 beta in the corneal tissue of the treated group are obviously increased. Tumor necrosis factor alpha, interleukin 6 and interleukin 1 beta expression are upregulated after in vivo injury of corneal tissue. The detection result shows that the cell inducer and the low-temperature freezing treatment can cause inflammatory stress reaction of the treated corneal tissue, so that the signal of simulating corneal injury in vivo is achieved, and the limbal stem cells are activated.
The results of paraffin embedding, sectioning and immunofluorescent staining of fresh and treated corneas are shown in fig. 5, respectively. The results showed that epithelial structures of both corneal tissues were preserved intact, and limbal stem cells with positive expression of P63 and negative expression of K3 were seen at the basal portion of the limbal position in both fresh cornea and treated corneal tissues. The results show that the stem cell activity of the limbal stem cells in the tissue is not affected by the cell inducer and the low-temperature freezing treatment.
Flow cytometry detection was performed on two groups of primary cultured limbal stem cells, and the flow cytometry detection results are shown in fig. 6. The results show that the stem cell marker P63 of the fresh corneal group primary culture limbal stem cells has a positive cell rate of 62 percent and the corneal epithelial cell differentiation marker K3 has a positive cell rate of 32 percent. In the treatment group, the positive cell rate of P63 was increased to 91%, and the positive cell rate of K3 was decreased to 20%. The detection result shows that more limbal stem cells with better proliferation activity and lower differentiation degree can be obtained by the corneal tissue which is subjected to the cell inducer and the low-temperature freezing treatment.
The results show that the limbal stem cells obtained by the method for screening, storing and culturing the limbal stem cells have high proportion and good stem cell activity.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for producing a limbal stem cell, comprising the steps of:
obtaining corneal tissue, and pretreating the corneal tissue to be used;
adding a cell cryopreservation solution containing a cell inducer into the pretreated corneal tissue;
freezing the corneal tissue with the cell freezing solution;
the cell inducer is an agent that induces local inflammation or stress in cells in the corneal tissue, but does not directly cause cell death;
the cell freezing medium is a mixed solution of a cell culture medium containing the cell inducer and a cryoprotectant;
the cell inducer is prednisolone acetate and staurosporine, the concentration of the prednisolone acetate in the cell culture solution is 4 mu g/ml, and the concentration of the staurosporine in the cell culture solution is 20 ng/ml; or the cell inducer is prednisolone acetate and tumor necrosis factor alpha, the concentration of the prednisolone acetate in the cell culture solution is 4 mug/ml, and the concentration of the tumor necrosis factor alpha in the cell culture solution is 10 ng/ml;
freezing by adopting liquid nitrogen;
the freezing time is 3 months or 6 months.
2. The method for producing limbal stem cells according to claim 1, wherein the volume ratio of the cryoprotectant to the cell culture solution containing the cell inducer is 1: 2 to 20.
3. The method for producing limbal stem cells according to claim 2, wherein the volume ratio of the cryoprotectant to the cell culture solution containing the cell inducer is 1: 2 to 10.
4. The method of claim 1, wherein the cryoprotectant is selected from the group consisting of one or more of dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide, methanol, polyvinylpyrrolidone, fructose, sucrose, dextran, albumin, polyethylene glycol, ethyl starch, and egg yolk.
5. The method of claim 1, wherein the pre-treating comprises: using physiological buffer solution containing or not containing antibiotics to carry out conventional cleaning, disinfection and separation on the corneal tissue to be used; and/or the presence of a catalyst in the reaction mixture,
the corneal tissue is selected from any one or more of human, rodent and other mammal corneal tissue, and is any one of fresh corneal tissue or corneal tissue subjected to short-term preservation, wherein the short-term preservation time is 1-20 days.
6. The method of claim 5, wherein the isolated culture is performed by one or more methods selected from the group consisting of tissue adherence and digestion.
7. The method of claim 6, wherein the tissue adherence method comprises the steps of: after the limbal tissue is separated from the corneal tissue, the limbal tissue epithelial layer is attached to a culture vessel and cultured, and the limbal stem cells are migrated from the limbal tissue to obtain limbal stem cells.
8. The method of claim 6, wherein the digestion comprises the steps of: the corneal epithelial layer containing the limbal stem cells is digested and separated from corneal tissue using a digestive enzyme, and then the corneal epithelial layer is inoculated into a culture vessel and cultured to obtain the limbal stem cells.
9. The method of claim 1, further comprising the steps of: and (5) separating and culturing the frozen corneal tissue to obtain the limbal stem cells.
10. The method for producing limbal stem cells according to claim 9, wherein the cell-inducing agent is added to the cell culture medium used for the culture in an amount of 1ng/ml to 100. mu.g/ml.
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