阅读说明：本技术 一种无血清培养角膜缘基质干细胞并于体外诱导成球和诱导分化的方法 (Method for culturing limbal stromal stem cells without serum and inducing balling and differentiation in vitro ) 是由 欧阳宏 朱丽琼 于 2018-06-06 设计创作，主要内容包括：本发明公开了一种无血清培养角膜缘基质干细胞及其诱导分化和干细胞成球的方法。本发明还同时提供了用于体外诱导角膜缘基质干细胞成球或分化成角膜缘基质细胞的培养基组合。本发明所使用的无血清培养基组合,能提供细胞生长增殖所需的充足营养与良好环境,能够稳定的在体外扩增培养角膜缘基质干细胞,而且能确保扩增培养后仍保持角膜缘基质干细胞的干性以及特异性。同时成功构建了诱导分化成角膜基质细胞的体系,可用于角膜缘基质干细胞的实验研究、角膜病变的细胞治疗以及角膜损伤的移植,具有广泛的科学效益和社会经济效益。(The invention discloses a method for culturing limbal stromal stem cells without serum and inducing differentiation and stem cell balling of the limbal stromal stem cells. The invention also provides a culture medium combination for inducing the limbal stromal cells to be spheronized or differentiated into the limbal stromal cells in vitro. The serum-free culture medium combination used by the invention can provide sufficient nutrition and good environment required by cell growth and proliferation, can stably amplify and culture the limbal stromal stem cells in vitro, and can ensure that the dryness and specificity of the limbal stromal stem cells are still maintained after the amplification culture. And meanwhile, a system for inducing differentiation into corneal stromal cells is successfully constructed, the system can be used for experimental research of corneal limbal stromal stem cells, cell treatment of corneal pathological changes and transplantation of corneal injury, and has wide scientific benefits and social and economic benefits.)

1. limbal stromal stem cellA culture medium comprising a basal medium and an additive component; wherein the additive components comprise: 50-150 IU of penicillin, 90-110 mug/mL of streptomycin, 40-60 mug/mL of gentamycin, ITS, 5-30 ng/mL of human recombinant EGF, 0.05-0.2 mM of L-ascorbic acid-2-phosphate, 1 x 10^-6～1×10^-10M dexamethasone and 50-200 ng/mL cholera toxin.

2. A limbal stromal cell balling culture medium is characterized by comprising a basic culture medium and an additive component; wherein the additive components comprise: 50-150 IU of penicillin, 90-110 mug/mL of streptomycin, 40-60 mug/mL of gentamycin, 5-30 ng/mL of human recombinant EGF, 0.5-4 mM of L-glutamine, 0.5-5% of B27 and ITS.

3. A limbal stromal cell culture medium comprising a basal medium and additional components; wherein the additive components comprise: 50-150 IU of penicillin, 90-110 mug/mL of streptomycin, 40-60 mug/mL of gentamycin, ITS, 0.2-2 mM of L-ascorbic acid-2-phosphate and 10-200 ng/mL of human recombinant FGF 2.

4. The culture medium according to any one of claims 1 to 3, wherein the basal medium is low-sugar DMEM, MCDB-201 and/or Advanced DMEM.

5. A culture medium combination for inducing spheronization and differentiation in vitro of a limbal stromal stem cell cultured in a serum-free manner, wherein the culture medium combination is the limbal stromal stem cell culture medium according to claim 1 and the limbal stromal stem cell spheronization culture medium according to claim 2, or the culture medium combination is the limbal stromal stem cell culture medium according to claim 1 and the limbal stromal cell culture medium according to claim 3.

6. A method for culturing serum-free limbal stromal stem cells in vitro, which is characterized in that after cleaning and cutting limbal tissue, the limbal tissue is subjected to enzymolysis, and the product after the enzymolysis is placed in the limbal stromal stem cell culture medium of claim 1 for culture.

7. An in vitro culture method for limbal stromal cells balling is characterized in that limbal stromal cells are cultured to the generation P1-P5, washed and digested, and the digested product is placed in the limbal stromal cell balling culture medium of claim 2 for culture.

8. An in vitro culture method of corneal limbal stromal cells is characterized in that the corneal limbal stromal cells are cultured to the generations from P1 to P5, and after being cleaned and digested, the digested product is firstly placed in the corneal limbal stromal cell culture medium of claim 1 for culture; after 12-36 h of culture, the cells are cultured in the corneal limbal stromal cell culture medium of claim 3.

9. A method for culturing limbal stromal cells without serum and inducing the limbal stromal cells to form balls in vitro is characterized by comprising the steps of culturing the limbal stromal cells without serum in vitro and culturing the limbal stromal cells to form balls in vitro, and the method comprises the following specific steps: firstly, culturing the limbal stromal stem cells by the method of claim 6 until the generation is from P1 to P5, washing, digesting, and culturing the digested product in the limbal stromal stem cell spherulization culture medium of claim 2.

10. A method for culturing corneal limbal stromal cells in a serum-free manner and inducing differentiation in vitro is characterized by comprising the steps of culturing the corneal limbal stromal cells in vitro in the serum-free manner and culturing the corneal limbal stromal cells in vitro, and comprises the following specific steps: firstly, culturing the limbal stromal stem cells by the method of claim 6 until the cells reach the generation P1-P5, washing and digesting the cells, and then putting the digested products into the limbal stromal stem cell culture medium of claim 1 for culturing; after 12-36 h of culture, the cells are cultured in the corneal limbal stromal cell culture medium of claim 3.

Technical Field

The invention belongs to the technical field of cell engineering. More particularly, it relates to a serum-free culture method of limbal stromal cells, and a culture method of limbal stromal cells induced in vitro spheronization and induced in vitro differentiation.

Background

The cornea is a layer of transparent tissue present at the front end of the eyeball and has a certain radius of curvature in space. Physiologically, the cornea is divided into a central transparent corneal zone and a peripheral limbal zone. The central transparent cornea area is divided into five layers, namely a corneal epithelial layer, a front elastic layer, a matrix layer, a back elastic layer and an endothelial cell layer from outside to inside. Wherein the stroma accounts for 90% of the corneal thickness and is mainly composed of type I collagen, type V collagen, adhesive and corneal stromal cells. Corneal stromal cells are derived from neural crest cells in a developmental manner, and after birth, these cells are no longer proliferating and dividing and are usually quiescent. These corneal stromal cells can maintain the integrity and transparency of the cornea by secreting ECM. When the cornea is damaged, or after the corneal stroma is damaged due to a series of diseases such as bacterial fungal keratitis and alkali burn, leucoma can be caused, when the leucoma appears, an opaque area can be caused to appear on the cornea, the vision of a patient is seriously affected, and an effective treatment means is not available at present. The traditional treatment method of the leucoma is mainly corneal transplantation. However, corneal transplantation has a series of problems, such as insufficient donor source, postoperative rejection, various complications and the like, and the clinical treatment effect is not ideal. Therefore, the treatment of leukoplakia lesions by transplantation of limbal stromal cells cultured in vitro has become a focus of attention in recent years.

For the culture of the corneal limbal stromal stem cells, the current method mainly adopts an enzyme digestion method to obtain primary cells from the corneal limbal tissue and then carries out in vitro amplification culture. How to maintain the dryness and characteristics of the stromal stem cells after in vitro culture is very important and is a problem to be solved urgently in cell engineering.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a serum-free culture method of the limbal stromal stem cells, which ensures that the dryness and specificity of the limbal stromal stem cells are still maintained after in vitro amplification culture. Ensures that after the corneal stromal cells cultured in vitro are transplanted back to the body for treatment, the corneal stromal cells can be differentiated into the corneal stromal cells for damage repair so as to treat corneal leukoplakia and other corneal lesions, thereby creating conditions for further clinical transplantation.

The first purpose of the invention is to provide a culture medium of the corneal limbal stromal stem cells.

the second purpose of the invention is to provide a culture medium for the limbal stromal stem cell balling.

the third purpose of the invention is to provide a culture medium of the corneal limbal stromal cells.

The fourth purpose of the invention is to provide a culture medium for inducing the spheronization or differentiation of the limbal stromal stem cells into the limbal stromal cells in vitro.

The fifth object of the present invention is to provide a culture method for obtaining serum-free in vitro corneal limbal stromal stem cells using the above-mentioned culture medium for corneal limbal stromal stem cells.

the sixth purpose of the invention is to provide a culture method for inducing the limbal stromal cells to form the spheroids in vitro, which comprises the following steps: and an in vitro culture method for obtaining the limbal stromal cell spheroids by using the culture medium for the limbal stromal cell and the culture medium for the limbal stromal cell spheroids.

The seventh purpose of the invention is to provide a culture method for inducing the differentiation of the corneal limbal stromal stem cells into the corneal stromal cells in vitro, which comprises the following steps: an in vitro culture method for obtaining a limbal stromal cell using the culture medium for a limbal stromal cell and the culture medium for a limbal stromal cell.

The above purpose of the invention is realized by the following technical scheme:

the invention relates to a limbal stromal cell culture medium, which comprises a basal culture medium and an additive component; wherein the additive components comprise: 50-150 IU of penicillin, 90-110 mug/mL of streptomycin, 40-60 mug/mL of gentamycin, ITS, 5-30 ng/mL of human recombinant EGF, 0.05-0.2 mM of L-ascorbic acid-2-phosphate (ascorbyl acid-2-phosphate), and 1 x 10^-6～1×10^-10M dexamethasone and 50-200 ng/mL cholera toxin.

In the present invention, the basic medium refers to a serum-free medium capable of providing necessary nutrients to the limbal stromal cells or the corneal limbal stromal cells, and includes all the media known in the field of limbal stromal cell culture and suitable for culturing the limbal stromal cells or the corneal limbal stromal cells.

Preferably, the basic medium of the present invention is low-sugar DMEM, MCDB-201 and/or Advanced DMEM.

more preferably, the basal medium in the limbal stromal stem cell medium is low-carbohydrate DMEM and/or MCDB-201.

Preferably, the concentration of each of the additional components in the medium is: 100 IU of penicillin, 100 μ g/mL of streptomycin, 50 μ g/mL of gentamicin, ITS, 20 ng/mL of human recombinant EGF, 0.1 mM of L-ascorbic acid-2-phosphate (ascorbyl acid-2-phosphate), 1 × 10^-8 M dexamethasone and 100ng/mL cholera toxin.

More preferably, the formula ratio of the limbal stromal cell culture medium is as follows: every 500 mL of medium contained: 5 mL of 10 XPicillin-streptomycin double antibody solution, 500 μ L gentamycin, 5 mL of ITS, 1 mL of human recombinant EGF, 1 mL of L-ascorbic acid-2-phosphate, 1 mL of cholera toxin, 288 mL of low-sugar DMEM, and the balance MCDB-201.

The invention also relates to a limbal stromal cell balling culture medium, which comprises a basal culture medium and an additive component; wherein the additive components comprise: 50-150 IU of penicillin, 90-110 mug/mL of streptomycin, 40-60 mug/mL of gentamycin, 5-30 ng/mL of human recombinant EGF, 0.5-4 mM of L-glutamine, 0.5-5% of B27 and ITS.

Preferably, the basal medium in the limbal stromal stem cell spheronization medium is Advanced DMEM.

Preferably, the concentration of each of the additional components in the medium is: 100 IU of penicillin, 100 mug/mL of streptomycin, 50 mug/mL of gentamicin, 20 ng/mL of human recombinant EGF, 2mM of L-glutamine, 2% of B27 and ITS.

more preferably, the formula proportion of the limbal stromal cell spheration culture medium is as follows: every 500 mL of medium contained: 5 mL of 10 XPicillin-streptomycin double-antibody solution, 500 μ L gentamicin, 5 mL of ITS, 1 mL of human recombinant EGF, 5 mL of L-glutamine and 5 mL of B27, with the balance being Advanced DMEM.

The invention also relates to a limbal stromal cell culture medium, which comprises a basal medium and an additive component; wherein the additive components comprise: 50-150 IU of penicillin, 90-110 mug/mL of streptomycin, 40-60 mug/mL of gentamycin, ITS, 0.2-2 mM of L-ascorbic acid-2-phosphate and 10-200 ng/mL of human recombinant FGF 2.

Preferably, the basal medium in the limbal stromal cell culture medium is low-carbohydrate DMEM.

Preferably, the concentration of each of the additional components in the medium is: 100 IU of penicillin, 100 μ g/mL of streptomycin, 50 μ g/mL of gentamicin, ITS, 1 mM of L-ascorbic acid-2-phosphate (ascorbyl acid-2-phosphate), and 100ng/mL of human recombinant FGF 2.

More preferably, the corneal limbal stromal cell culture medium has the following formula ratio: every 500 mL of medium contained: 5 mL of 10 XPicillin-streptomycin double-antibody solution, 500 mu L of gentamycin, 5 mL of ITS, 1 mL of L-ascorbic acid-2-phosphate and 1 mL of human recombinant FGF2, and the balance of low-sugar DMEM.

In the present invention, the ITS refers to Insulin, Transferrin, Selenium Solution. The ITS of the invention is purchased from Gbico ITS reagent, is 100X solution, and can be used after being diluted in proportion.

in the invention, the B27 refers to a B27 additive; the B27 additive is serum-free additive; the B27 additive of the invention is available from Gbico under the accession number 12587010.

The invention also relates to a culture medium combination for culturing the limbal stromal cells without serum and inducing the spheronization and differentiation in vitro, which is the combination of the culture medium for the limbal stromal cells and the culture medium for the limbal stromal cells to spheronize or the combination of the culture medium for the limbal stromal cells and the culture medium for the limbal stromal cells.

Specifically, the culture medium for inducing the limbal stromal cell to sphere in vitro comprises the limbal stromal cell culture medium and the limbal stromal cell sphere culture medium.

Specifically, the culture medium for inducing the differentiation of the limbal stromal cells into the limbal stromal cells in vitro comprises the limbal stromal cell culture medium and the limbal stromal cell culture medium.

the culture medium provided by the invention is a serum-free culture medium with determined chemical components, has stable quality and strong batch controllability, is easy to popularize and market, and is an ideal culture medium for the basic research and the clinical application research of the limbal stromal cells and the limbal stromal cells.

The invention also relates to a serum-free in vitro culture method of the limbal stromal cells, which comprises the steps of cleaning and cutting limbal tissues, carrying out enzymolysis on the limbal tissues, and placing products after the enzymolysis into the limbal stromal cell culture medium for culture.

Preferably, the enzymatic hydrolysis is carried out with collagen type L protease.

Preferably, the concentration of the L-type collagenase is 0.1-1 mg/mL, and the enzymolysis time is 3-12 h.

Preferably, Matrigel is added before the product after enzymolysis is placed in the corneal limbal stromal cell culture medium for culture.

More preferably, the Matrigel is added, followed by incubation at 37 ℃ for 10 min, and the isolated limbal stromal stem cells are added.

The invention also relates to an in vitro culture method of the limbal stromal cell spheroids, which comprises the steps of culturing the limbal stromal cells to the generation P1-P5, cleaning, digesting, and placing the digested product in the limbal stromal cell spheroids culture medium for culture.

preferably, the washing is washing with PBS; the digestion is carried out by 0.01 to 0.25 percent of pancreatin containing EDTA.

Preferably, the culture plate is coated with Poly-HEMA 2 days before the digested product is cultured in the limbal stromal stem cell sphering medium.

More preferably, the culture plate is coated with Poly-HEMA, incubated at 37 ℃ for 2-14 days, and then the isolated limbal stromal stem cells are added.

the invention also relates to an in vitro culture method of the limbal stromal cells, which comprises the steps of culturing the limbal stromal cells to P1-P5 generations, cleaning and digesting, and placing digested products into the limbal stromal cell culture medium for culture; and after 12-36 h of culture, changing the culture medium into the limbal stromal cell culture medium for culture.

The invention also relates to a method for culturing the limbal stromal stem cells without serum and inducing the limbal stromal stem cells to form spheres in vitro, which comprises the steps of culturing the limbal stromal stem cells without serum in vitro and culturing the limbal stromal stem cells to form spheres in vitro, and the method comprises the following steps: firstly, culturing the limbal stromal stem cells by adopting the method until the limbal stromal stem cells are cultured to P1-P5 generations, cleaning and digesting the limbal stromal stem cells, and placing the digested product into the limbal stromal stem cell spherulization culture medium for culturing.

The invention also relates to a method for culturing the limbal stromal cells without serum and inducing differentiation in vitro, which comprises the steps of culturing the limbal stromal cells without serum in vitro and culturing the limbal stromal cells in vitro, and the specific steps are as follows: firstly, culturing the limbal stromal stem cells by adopting the method until the cells are cultured to P1-P5 generations, cleaning and digesting the cells, and then putting the digested product into the limbal stromal stem cell culture medium for culturing; and after 12-36 h of culture, changing the culture medium into the limbal stromal cell culture medium for culture.

As a preferred technical scheme, the in vitro culture method of the serum-free limbal stromal stem cells comprises the following steps:

S11, cleaning the corneal limbus tissue by DMEM, and then cutting up the corneal limbus tissue for enzymolysis; the enzyme used is L-type collagenase with the concentration of 0.1-1 mg/mL, and is subjected to enzymolysis at 37 ℃ for 3-12 hours; filtering by using a 40 mu m cell sieve to remove residual tissue blocks; adding the Trypsin inhibitor with the same volume of 10mg/mL to terminate enzymolysis, centrifuging at 1500 rpm for 5 min, and removing the supernatant;

s12, resuspending the limbal stromal cells obtained by enzymolysis and centrifugation by using the limbal stromal cell culture medium;

S13, spreading the 10% Matrigel solution in a 24-well plate, standing at 37 ℃ for 10 min, and sucking the rest Matrigel solution;

S14, the resuspended limbal stromal stem cells were seeded into 10% Matrigel-plated 24-well plates at 37 ℃ in 5% CO₂And (5) culturing for 12-24 hours under the condition to adhere to the wall, and changing the culture solution every other day.

As a preferred technical scheme, the method for culturing the limbal stromal stem cells without serum and inducing the spheronization in vitro comprises the following steps:

S21, when the generation cells of the limbal stromal stem cells P1-P3 cultured in the step S14 grow to 80% -90% confluency, absorbing and discarding the liquid, washing with PBS once, digesting with 0.01% -0.25% pancreatin containing EDTA, adding equal volume of 10mg/mL Trypsin inhibitor to terminate enzymolysis, centrifuging at 1500 rpm for 5 min, discarding the supernatant;

s22, re-suspending the limbal stromal cells obtained by enzymolysis and centrifugation by using the limbal stromal cell balling culture medium;

S23, dissolving the mixture by using a 120 mg/mL Poly-HEMA solution overnight at 37 ℃ in a shaking table, spreading the mixture in a 12-hole plate, and standing the mixture for 48 hours at 37 ℃ for use;

S24, planting the resuspended corneal limbal stromal stem cells in a 12-well plate plated with 120 mg/mL Poly-HEMA solution at 37 deg.C with 5% CO₂culturing under the condition, and observing the cell state every other day;

S25, changing the culture solution by using the limbal stromal cell balling culture solution every day, and culturing for about 7 days.

as a preferred technical scheme, the method for serum-free culturing of the limbal stromal stem cells and in vitro differentiation induction comprises the following steps:

S31, when the generation cells of the limbal stromal stem cells P2-P4 cultured in the step S14 grow to 80% -90% confluency, absorbing and discarding the liquid, washing with PBS once, digesting with 0.01% -0.25% pancreatin containing EDTA, adding equal volume of 10mg/mL Trypsin inhibitor to terminate enzymolysis, centrifuging at 1500 rpm for 5 min, discarding the supernatant;

s32, resuspending the limbal stromal cells obtained by enzymolysis and centrifugation by using the limbal stromal cell culture medium;

S33, spreading the 10% Matrigel solution in a 24-hole plate, placing the plate in a 37 ℃ incubator, standing for 10 min, and then sucking the rest Matrigel solution;

S34, planting the resuspended corneal limbal stromal stem cells in a 24-well plate plated with 10% Matrigel, and placing at 37 ℃ with 5% CO₂culturing in an incubator;

S35, changing the culture solution of the limbal stromal cells every other day to perform induced differentiation culture, and observing the growth state of the cells in a microscope;

S36, changing the culture solution every 2 days by using the culture solution of the limbal stromal cells, and carrying out differentiation culture for about 7 days.

Compared with the prior art, the invention has the following beneficial effects:

The serum-free culture medium used by the invention can stably amplify and culture the limbal stromal stem cells in vitro, construct a culture system for balling the stem cells in vitro and express a corresponding stem cell Marker, and experiments prove that the limbal stromal stem cells cultured in vitro have dryness; meanwhile, a system for inducing differentiation into the corneal limbal stromal cells is successfully constructed, and a specific Marker of the corneal limbal stromal cells can be specifically and highly expressed, which fully indicates that the differentiation system is successfully constructed; compared with other culture methods containing serum, the method avoids the influence of serum components on experimental research, more clearly realizes the effect of each component in the culture solution on corneal stroma cell culture, improves the repeatability of cell culture and experimental results, is easier for purifying cell products, is beneficial to industrialization, and has wide application and development prospects.

Drawings

FIG. 1 is a cell morphology diagram of corneal limbal stromal stem cells cultured without serum.

FIG. 2 is a diagram of the cell morphology of limbal stromal stem cells in spheronization culture.

FIG. 3 is a cell morphology map of limbal stromal cells.

FIG. 4 is a graph showing the QPCR results for the specific marker KERA for limbal stromal cells (limbal stromal cells cultured in serum-free induction according to the invention are labeled keratocytes, whereas limbal stromal cells cultured in serum-containing medium are labeled fibroplasts).

FIG. 5 is a graph showing the result of QPCR of ALDH3A1, a marker specific to limbal stromal cells (limbal stromal cells cultured in a serum-free induction medium according to the present invention are labeled as keratocytes, whereas limbal stromal cells cultured in a serum-containing medium are labeled as fibroplasts).

FIG. 6 is a graph showing the QPCR results for fibroblast-specific marker α SMA (limbal stromal cells cultured in serum-free induction according to the present invention are labeled keratocytes, whereas limbal stromal cells cultured in serum-containing medium are labeled fibroplasts).

FIG. 7 is a graph showing the QPCR results for specific marker FN for Fibroblasts (the limbal stromal cells cultured in the serum-free induction culture according to the present invention are labeled keratocytes, while the limbal stromal cells cultured in the serum-containing culture medium are labeled fibroplasts).

FIG. 8 is a cell morphology of limbal stromal cells cultured in serum-containing medium.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. It is within the scope of the present invention to make simple modifications or alterations to the methods, procedures or conditions of the present invention without departing from the spirit and substance of the invention; unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

the ITS was purchased from Gbico ITS reagent as a 100X solution, diluted in proportion for use.