CN106916787B - A kind of limbal stem cell culture medium and its cultural method - Google Patents
A kind of limbal stem cell culture medium and its cultural method Download PDFInfo
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Abstract
The invention discloses a kind of limbal stem cell culture medium and its cultural method, nutrient media components are as follows: 5mL 100 × dual anti-, 10~20ng/ml people recombinate EGF, 5~10ug/ml insulin, 1~5x10‑9M 3- iodine thyronine, 0.2~1ug/ml hydrocortisone, 10~20ng/ml cholera toxin, 10~15% fetal calf serums, surplus are DMEM and/or DMEM/F12；Coating treatment is done to materials such as culture plates using the substrate that I-type collagen is grown as limbal stem cell, improve the purity and stability of limbal stem cell, using culture medium isolated cornea limbal stem cell provided by the invention, p63, Pax6 antibody positive rate are 96%~100% in cell.A kind of non-trophoblast, DNAcarrier free limbal stem cell cultural method are established, provides stable cell origin for the research of limbal stem cell specific mechanism and transplantation treatment.
Description
Technical field
The present invention relates to technical field of stem cell culture, more particularly, to a kind of limbal stem cell culture medium and its
Cultural method.
Background technique
Limbal stem cell is cornea and conjunctiva, sclera junction section, and the mark with the identification of cornea is BowmanShi film
Termination；Goblet cell is free from the identification marker of conjunctiva, the wide about 1~2mm of limbal stem cell, only epithelial layer herein
And hypothallus, epithelium layer contain 10 confluent monolayer cells, irregular arrangement, cell is in small cylindric, nuclear hyperchromatism.Its deep matrix
Cell is one layer of roundlet column or cuboid cell, and nucleus is oval, parallel with surface, is formed in basal part nipple, shape
At special " fence " sample epithelial structure, wherein containing pigment and rete vasculosum abundant, and contacted closely with basilar memebrane.On cornea
Skin is the non-cornified cell of single layer of ordered arrangement, and the update of corneal epithelial cell derives from limbal stem cell (LSCs), right
The maintenance of corneal transparency, eyesight plays an important role.When renewal process carries out, limbal stem cell is migrated to Central corneal, is gone forward side by side
Row differentiation, this complete process probably need 14 days.LSCs shortage is worldwide, catastrophic ophthalmology difficult and complicated illness, seriously
Human vision quality is threatened, lacks effective treatment means at present.Common cause includes eye traumas, alkali burn and immunity eye disease
Deng clinically, showing as cornea opacification and ablepsia；Eye examination with conjunctiva on cornea, occur new vessels,
Scar and atretoblepharia are formed as diagnostic criteria.
The treatment means traditional for limbal stem cell deficiency include amnion transplantation and LSCs transplanting, but amnion transplantation can
Lead to corneal epithelium phenotypic alternation, and traditional LSCs transplanting needs that tissue is more, iatrogenic injury is larger, clinical therapeutic efficacy is equal
It is undesirable.Autologous corneal limbus tissue transplantation is not suitable for the patient of cornea of both eyes edge lesion.There is row in allogeneic cornea edge tissue transplantation
Reprimand reaction.Therefore, the serious keratonosus of Penetrating Keratoplasty for Treatment is combined using the limbal stem cell transplantation of in vitro culture
Become, becomes hot spot concerned by people in recent years.
Culture for limbal stem cell, method mainly uses a variety of different basal layers such as source of mouse NIH 373 at present
Trophoderm, source of people amnion, Fibrin Glue, Myogel, plasma polymer coating, people's recombinant collagen substrate etc. are to corneal limbus
Stem cell is cultivated.These methods can obtain epithelium and plant piece, and be used successfully to the supplement of the stem cell lacks to treat eye
Surface diseases.
Though the cultural method of existing limbal stem cell can obtain epithelial cell, since 373 cell of NIH is external non-
Human archeocyte easily causes the propagation of immune response and the derivative pathogen of source of mouse；And source of people amnion is obtained due to being difficult to, and is expended
Greatly, it needs further to detect whether containing HIV, hepatitis virus etc., and there are opaque, has the shortcomings that certain thickness, be easy
It is integrated with corneal stroma, has certain influence to corneal thickness and structure.It is dry that above-mentioned factor can all influence the corneal limbus that culture obtains
Eye is to light and visual sensitivity after the transfer for cell, and existing method is only used for the disposable of limbal stem cell,
Limbal stem cell can not be carried out stablizing isolation and culture.
Summary of the invention
The technical problem to be solved by the present invention is to overcome drawbacks described above of the existing technology, a kind of novel angle is provided
The culture medium of film limbal stem cell.
A second object of the present invention is to provide a kind of cultural methods of limbal stem cell.
The purpose of the present invention is what is be achieved by the following technical programs:
A kind of limbal stem cell culture medium contains following components in culture medium: 5mL 100 × dual anti-, 10~20ng/ml
People recombinates EGF, 5~10ug/ml insulin, 1~5x10-9M 3- iodine thyronine, 0.2~1ug/ml hydrocortisone,
10~20ng/ml cholera toxin, 10~15% fetal calf serums, surplus are DMEM and/or DMEM/F12.
The present invention establishes novel culture system, with epidermal growth factor EGF, insulin, 3,3 ', 5-Triiodo-L-
Thyronine (3- iodine thyronine), hydrocortisone, cholera toxin etc. can promote the factors such as LSCs proliferation as culture
Base component can significantly improve the purity and quantity of limbal stem cell.
It is after cleaning corneal limbal tissue, diagonally the present invention also provides a kind of isolated culture method of limbal stem cell
Film edge tissue carries out digesting to obtain limbal stem cell, and above-mentioned limbal stem cell culture medium is added and is cultivated；The enzyme
Solution is primary enzymolysis either secondary enzymolysis；The secondary enzymolysis is first to be digested with IV clostridiopetidase A, then digested with alkali protease.
The primary enzymolysis of corneal limbal tissue of the present invention can use traditional mode of action, such as utilize complex enzyme enzyme
Solution, or trypsin digestion is used merely, the enzymolysis time of the trypsase is 10min~20min.
Specifically, the isolated culture method of limbal stem cell of the present invention is: corneal limbal tissue is taken, using containing dual anti-
PBS cleaning after, digested, after the product after enzymatic hydrolysis is cultivated with the culture medium of limbal stem cell, passage.
IV clostridiopetidase A and the separation of trypsase secondary enzymolysis can be used in the present invention, makes corneal limbal tissue under secondary enzymolysis
Sufficiently enzymatic hydrolysis increases cell yield to guarantee to obtain individual cells, reduce the damage to cell.
Preferably, when using secondary enzymolysis, the concentration of the IV clostridiopetidase A is 0.2~0.3%；The IV collagenase digestion
In DMEM/F12；The enzymolysis time of the IV clostridiopetidase A is 2~3h；The enzymolysis time of the trypsase is 10~20min.
It preferably, in the isolated culture method of above-mentioned limbal stem cell, is cultivated in addition limbal stem cell
Before, I-type collagen is first added.
Specifically, the limbal stem cell isolated culture method the following steps are included:
S1. corneal limbal tissue is taken, after containing dual anti-PBS cleaning, first 2h is digested with 0.2%IV collagen, removes collagen
0.25% trypsin solution enzymolysis, digestion 10min~20min is added in enzyme solution, and the DMEM containing FBS is added and terminates enzymolysis, digestion,
Centrifugation removal supernatant must separate after limbal stem cell；
S2. 10%I collagen type is placed in ice bath, after culture dish addition I-type collagen is coated with, is added
The culture medium of limbal stem cell after separation is resuspended cell and is cultivated.
Compared with prior art, the invention has the following advantages:
Present invention firstly provides the culture medium of limbal stem cell, contain following components in culture medium:
5mL 100 × dual anti-, 10~20ng/ml people recombinate EGF, 5~10ug/ml insulin, 1~5x10-9M 3- iodine first
Shape gland original ammonia acid, 0.2~1ug/ml hydrocortisone, 10~20ng/ml cholera toxin, 10~15% fetal calf serums, surplus are
DMEM and/or DMEM/F12；Each component is optimized, the low differentiation state of limbal stem cell can be preferably maintained, this
Invention does coating treatment to materials such as culture plates using the substrate that I-type collagen is grown as limbal stem cell, mentions
The high purity and stability of limbal stem cell, is promoted the growth of limbal stem cell with fetal calf serum, is capable of selectivity
Isolated cornea limbal stem cell, and be separately cultured resulting limbal stem cell purity and quantity it is all higher, stemness and proliferation energy
Power is good.Experiments have shown that using culture medium isolated cornea limbal stem cell provided by the invention, p63, Pax6 antibody positive in cell
Rate is 96%~100%.
The present invention establishes a kind of novel non-trophoblast, DNAcarrier free limbal stem cell cultural method, realize it is uniform,
The functional limbal stem cell of efficient amplification in vitro, provides the cell origin of fast and stable.It is dry thin to improve gained corneal limbus
The purity of born of the same parents improves the quality of gained limbal stem cell to establish cell bank as spare, is limbal stem cell specificity
Mechanism Study and transplantation treatment provide the cell origin of fast and stable.
Detailed description of the invention
Fig. 1 is carrier-free, non-trophoblast human limbal stem cell culture (cellular morphology under microscope).
Fig. 2 is limbal stem cell specificity marker immunocytochemical stain；Fig. 2A is that (green, corneal limbus are dry thin by P63
Born of the same parents' mark)；Fig. 2 B is PAX6 (red)；Fig. 2 C is after fusion (blue is DAP).
Fig. 3 be using behind described in embodiment 1 culture medium culture 12 days cellular morphology figure (cellular morphology under microscope, 10
×)。
Fig. 4 be using culture medium culture limbal stem cell described in comparative example 1 cellular morphology figure (form under microscope,
10×)。
Fig. 5 be using culture medium culture limbal stem cell described in comparative example 2 cellular morphology figure (form under microscope,
10×)。
Fig. 6 be using culture medium culture limbal stem cell described in comparative example 3 cellular morphology figure (form under microscope,
10×)。
Specific embodiment
The contents of the present invention are further illustrated with specific embodiment with reference to the accompanying drawings of the specification, but should not be construed as to this
The limitation of invention.Without departing from the spirit and substance of the case in the present invention, to simple made by the method for the present invention, step or condition
Modifications or substitutions all belong to the scope of the present invention；Unless otherwise specified, technological means used in embodiment is art technology
Conventional means known to personnel.
10%I collagen type solution: 1mL I-type collagen is dissolved in 9mL DMEM/F12 culture medium.
IV clostridiopetidase A: 0.2wt%IV collagenase solution is configured to DMEM/F12 culture medium.
People recombinates EGF stock solution: being prepared with PBS, is made into 5 μ g/mL EGF stock solutions.
Insulin stock liquid: with 0.005M HCl preparation, concentration 2.5g/L.
3,3 ', 5-Triiodo-L-Thyronine stock solution: 13.6mg 3,3 ', 5-Triiodo-L-Thyronine are molten
85mL PBS is added in 15mL0.02M NaOH in solution；It takes the prepared liquid of 0.1ml, adds PBS to 20ml, using as storage
Standby liquid, concentration are as follows: 10-6M。
Hydrocortisone stock solution: 5mg hydrocortisone is dissolved in 1mL dehydrated alcohol, and PBS to 25mL is added, and concentration is
200μg/mL。
Cholera toxin stock solution: 1mg cholera toxin is dissolved in 1mL steril cell culture grade pure water, is taken wherein 0.1mL, is added
Enter PBS to 20mL, concentration are as follows: 5ug/mL.
Embodiment 1
Limbal stem cell culture medium: 220mL DMEM, 220mL DMEM/F12,50mL FBS, 5mL 100 × dual anti-
(100IU penicillin, 100ug/ml streptomysin), 1mL people recombinate EGF stock solution, 1mL insulin stock liquid, 1mL3,3 ', 5-
Triiodo-L-Thyronine stock solution, 1mL hydrocortisone stock solution, 1mL cholera toxin stock solution are placed in 4 DEG C of guarantors
It deposits, uses preceding 37 DEG C of rewarmings.
Under surgical operation microscope, with tissue clamps and corneal scissors clip people's corneal limbal tissue with containing dual anti-in gnotobasis
The PBS of (Pen .- Strep, 1 ×) is rinsed 2 times, each 5min；Tissue is shredded with scissors again.
According to the volume of tissue block, every 1cm35ml 0.2wt%IV collagen enzyme solution is added in tissue block, and 37 DEG C of gentle agitations disappear
After changing 2h, IV collagen enzyme solution is removed, 0.25% trypsin solution of 5mL is added, after uniform suspension cell, with 100 μm of mesh screen mistakes
Filter, then 37 DEG C of further digestion 15min are placed in, the DMEM containing 10%FBS is added and terminates digestion, 1000rpm is centrifuged 5min, reject
Supernatant.
10%I collagen type is placed in ice bath, every hole is added 1mL 10%I collagen type and is wrapped in 6 orifice plates
Quilt is placed in 37 DEG C, contains 5%CO2It is incubated for 40min in incubator, is rinsed with PBS, removes PBS.
Postdigestive cell is resuspended in the limbal stem cell culture medium that 2mL rewarming is added, and is planted respectively in being coated with 10%I
In the hole of collagen type, 37 DEG C are placed in, contains 5%CO2It is cultivated in incubator, changes liquid every other day, and observe cell in microscope
Growth conditions.
Embodiment 2
Experimental method is with embodiment 1, and uniquely the difference is that, limbal stem cell culture medium used in the present embodiment forms
Are as follows: 204.5mL DMEM, 204.5mL DMEM/F12,75mL FBS, 5mL 100 × dual anti-(100IU penicillin, 100ug/ml
Streptomysin), 2mL people recombinates EGF stock solution, 2mL insulin stock liquid, 2.5mL 3,3 ', 5-Triiodo-L-Thyronine storage
Standby liquid, 2.5mL hydrocortisone stock solution, 2mL cholera toxin stock solution.
Comparative example 1
Experimental method is unique the difference is that concentration of the EGF in limbal stem cell culture medium is 2ug/ with embodiment 1
Ml, as a result, it has been found that cell slow growth, loses the form of epidermal cell, cell is in shuttle shape.
Comparative example 2
Experimental method is with embodiment 1, and uniquely the difference is that, serum-concentration is reduced to 2% (the i.e. additional amount of FBS from 10%
For 10mL), as a result, it has been found that the cell of cell becomes larger, nucleus becomes smaller, adherent ability difference of cell etc..
Comparative example 3
Experimental method is unique the difference is that concentration of the insulin in limbal stem cell culture medium is with embodiment 1
2ug/ml, as a result, it has been found that cell slow growth, the form of epidermal cell is unobvious, and cell is loose with Cell tracking, dead
Cell is more.
Claims (4)
1. a kind of isolated culture method of limbal stem cell, which is characterized in that be after cleaning corneal limbal tissue, to corneal limbus
Tissue carries out digesting to obtain limbal stem cell, and limbal stem cell culture medium is added and is cultivated；The enzymatic hydrolysis is secondary
Enzymatic hydrolysis；The secondary enzymolysis is first to be digested with IV clostridiopetidase A, then digested with alkali protease；
Wherein, contain following components in the limbal stem cell culture medium: 5mL 100 × dual anti-, 10-20ng/ml people's recombination
EGF, 5-10ug/ml insulin, 1-5 × 10-9M 3- iodine thyroid gland original nitronic acid, 0.2-lug/ml hydrocortisone, 10-20ng/
Ml cholera toxin, 10-15% fetal calf serum, surplus are DMEM and/or DMEM/F12.
2. the isolated culture method of limbal stem cell according to claim 1, which is characterized in that the IV clostridiopetidase A
Concentration is 0.2-0.3%.
3. the isolated culture method of limbal stem cell according to claim 1, which is characterized in that the IV clostridiopetidase A
Enzymolysis time is 2-3h.
4. the isolated culture method of limbal stem cell according to any one of claims 1 to 3, which is characterized in that adding
Enter before limbal stem cell cultivated, I-type collagen is first added.
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| CN109439628B (en) * | 2018-10-10 | 2021-09-24 | 中国海洋大学 | Primary culture method of limbal stem cells |
| CN109321527A (en) * | 2018-10-10 | 2019-02-12 | 中国海洋大学 | The extracorporeal culturing method of limbal stem cell stability |
| CN112126626B (en) * | 2020-09-30 | 2023-04-07 | 广东康盾创新产业集团股份公司 | Limbal stem cell culture medium and culture method |
| CN112626019A (en) * | 2020-12-28 | 2021-04-09 | 武汉爱尔眼科医院有限公司 | Preparation method of single cell suspension of cornea and corneal limbus |
| CN114540303A (en) * | 2022-02-21 | 2022-05-27 | 赛尔生命科技(深圳)有限公司 | Limbal stem cell culture solution and preparation method thereof |
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