CN106916787A - A kind of limbal stem cell culture medium and its cultural method - Google Patents
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Abstract
The invention discloses a kind of limbal stem cell culture medium and its cultural method, nutrient media components is：5mL 100 × dual anti-, 10~20ng/ml people restructuring EGF, 5~10ug/ml insulin, 1~5x10‑9The iodine thyronines of M 3,0.2~1ug/ml hydrocortisones, 10~20ng/ml cholera toxins, 10~15% hyclones, balance of DMEM and/or DMEM/F12；Coating treatment is done to materials such as culture plates as the substrate that limbal stem cell grows using I-type collagen, improve the purity and stability of limbal stem cell, the culture medium isolated cornea limbal stem cell provided using the present 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, the cell derived of stabilization is provided 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 technology
Limbal stem cell is cornea and conjunctiva, sclera junction section, is BowmanShi with the mark of the discriminating of cornea
The termination of film；Be free from goblet cell with the identification marker of conjunctiva, limbal stem cell about 1~2mm wide, only have herein on
Cortex and hypothallus, its epithelium layer contain 10 confluent monolayer cells, irregular arrangement, and cell is in small cylindric, nuclear hyperchromatism.Its deep
Stroma cell is one layer of roundlet column or cuboid cell, and nucleus is oval, parallel with surface, in basal part mammiform
Into, special " fence " sample epithelial structure is formed, wherein containing pigment and abundant rete vasculosum, and contacted closely with basilar memebrane.
Corneal epithelium is the non-cornified cell of individual layer of ordered arrangement, and the renewal of corneal epithelial cell derives from limbal stem cell
（LSCs）, corneal is transparent, eyesight maintenance plays an important role.When renewal process is carried out, limbal stem cell is to Central corneal
Migration, and broken up, this complete process probably needs 14 days.It is worldwide, catastrophic ophthalmology difficulty that LSCs lacks
Illness, it is serious to threaten human vision quality, effective treatment means are lacked at present.Common cause includes eye traumas, alkali burn and exempts from
Epidemic disease illness in eye etc., clinically, shows as cornea opacification and ablepsia；Eye examination is with conjunctiva, appearance on cornea
New vessels, scar and atretoblepharia are formed as diagnostic criteria.
The treatment means traditional for limbal stem cell deficiency include that amnion transplantation and LSCs are transplanted, but amnion transplantation can
Cause corneal epithelium phenotypic alternation, and traditional LSCs transplanting needs many, iatrogenic injuries of tissue are 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 the tissue transplantation of allogeneic cornea edge
Reprimand reaction.Therefore, using the keratonosus that the limbal stem cell transplantation joint Penetrating Keratoplasty for Treatment of in vitro culture is serious
Become, in recent years as focus of concern.
For the culture of limbal stem cell, current method mainly uses various different basalises such as mouse source NIH 373
The corneal edge such as trophoderm, people source amnion, Fibrin Glue, Myogel, plasma polymer coating, people's recombinant collagen substrate
Stem cell is cultivated.These methods can obtain epithelium and plant piece, and are used successfully to the supplement of the stem cell lacks and are used to treat eye
Surface diseases.
Though the cultural method of existing limbal stem cell can obtain epithelial cell, because the cells of NIH 373 are external non-
Human archeocyte, easily causes immune response and mouse source to derive the propagation of pathogen；And people source amnion is obtained due to being difficult to, expend
It is big, it is necessary to further detect whether containing HIV, hepatitis viruse etc., and exist it is opaque, with certain thickness shortcoming, easily
Integrated with corneal stroma, diagonal film thickness and structure have certain influence.Above-mentioned factor can all influence to cultivate the corneal limbus for obtaining and do
Cell after the transfer eye to light and visual sensitivity, and existing method is only used for the single use of limbal stem cell,
Cannot corneal limbal stem cell carry out stablizing separation and culture.
The content of the invention
The technical problems to be solved by the invention are the drawbacks described above for overcoming prior art to exist, there is provided a kind of new angle
The culture medium of film limbal stem cell.
Second object of the present invention is to provide a kind of cultural method of limbal stem cell.
The purpose of the present invention is 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
Restructuring EGF, 5~10ug/ml insulin, 1~5x10-9M 3- iodine thyronine, 0.2~1ug/ml hydrocortisones, 10
~20ng/ml cholera toxins, 10~15% hyclones, balance of DMEM and/or DMEM/F12.
The present invention establishes novel culture system, with EGF EGF, insulin, 3,3 ', 5-Triiodo-L-
Thyronine（3- iodine thyronines）, hydrocortisone, cholera toxin etc. the factor such as LSCs can be promoted to breed is used as culture
Base component, can significantly improve the purity and quantity of limbal stem cell.
The present invention also provides a kind of isolated culture method of limbal stem cell, is after corneal limbal tissue is cleaned, diagonally
Film edge tissue carries out digesting to obtain limbal stem cell, adds above-mentioned limbal stem cell culture medium to be cultivated；The enzyme
Solution is primary enzymolysis or secondary enzymolysis；The secondary enzymolysis are first to be digested with IV clostridiopetidase As, then are 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：Take the angle of human death fetus in 48h
Film edge tissue, after containing dual anti-PBS, is digested, by the product after enzymolysis with the culture medium of limbal stem cell
After being cultivated, passage.
The present invention can be separated using IV clostridiopetidase As and trypsase secondary enzymolysis, make corneal limbal tissue in secondary enzymolysis
Lower fully enzymolysis, so as to ensure to obtain the damage of individual cells, reduction to cell, increases cell yield.
Preferably, when using secondary enzymolysis, the concentration of the IV clostridiopetidase As is 0.2~0.3%；The IV collagenase digestions in
In DMEM/F12；The enzymolysis time of the IV clostridiopetidase As is 2~3h；The enzymolysis time of the trypsase is 10~20min.
Preferably, it is to add limbal stem cell to be cultivated in the isolated culture method of above-mentioned limbal stem cell
Before, I-type collagen is first added.
Specifically, the isolated culture method of the limbal stem cell is comprised the following steps：
S1. the corneal limbal tissue of human death fetus in 48h is taken, after containing dual anti-PBS, 0.2%IV collagens is first used
Enzymolysis 2h, removes collagen enzyme liquid, adds 0.25% trypsin solution enzymolysis, digestion 10min~20min, adds the DMEM containing FBS
Terminate enzymolysis, digestion, centrifugation removes the limbal stem cell after supernatant must be separated；
S2. 10% I-type collagen is placed in ice bath, culture dish is added after I-type collagen is coated with, is added point
The culture medium re-suspended cell of the limbal stem cell after is cultivated.
Compared with prior art, the invention has the advantages that：
Present invention firstly provides the culture medium of limbal stem cell, following components is contained in culture medium：
5mL 100 × dual anti-, 10~20ng/ml people restructuring EGF, 5~10ug/ml insulin, 1~5x10-9M 3- iodine first shapes
Gland original ammonia acid, 0.2~1ug/ml hydrocortisones, 10~20ng/ml cholera toxins, 10~15% hyclones, balance of DMEM
And/or DMEM/F12；Each component is optimized, the low differentiation state of limbal stem cell can be preferably maintained, the present invention
Coating treatment is done to materials such as culture plates as the substrate that limbal stem cell grows using I-type collagen, is improve
The purity and stability of limbal stem cell, the growth of limbal stem cell is promoted with hyclone, is capable of the separation of selectivity
Limbal stem cell, and be separately cultured gained limbal stem cell purity and quantity it is all higher, dryness and multiplication capacity are good
It is good.Experiment shows that, using the culture medium isolated cornea limbal stem cell of present invention offer, p63, Pax6 antibody positive rate are in cell
96%~100%.
The present invention establishes a kind of new non-trophoblast, DNAcarrier free limbal stem cell cultural method, realize it is homogeneous,
The functional limbal stem cell of efficient amplification in vitro, there is provided the cell derived of fast and stable.Improve gained corneal limbus dry thin
The purity of born of the same parents, improves the quality of gained limbal stem cell to set up cell bank as standby, is limbal stem cell specificity
Mechanism Study and transplantation treatment provide the cell derived of fast and stable.
Brief description of the drawings
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 P63（Green, corneal limbus is dry thin
Born of the same parents indicate）；Fig. 2 B are PAX6（It is red）；After Fig. 2 C are for fusion（Blueness is DAP）.
Fig. 3 is using the cellular morphology figure after medium culture described in embodiment 1 12 days（Cellular morphology under microscope, 10
×）.
Fig. 4 is the cellular morphology figure using medium culture limbal stem cell described in comparative example 1（Form under microscope,
10×）.
Fig. 5 is the cellular morphology figure using medium culture limbal stem cell described in comparative example 2（Form under microscope,
10×）.
Fig. 6 is the cellular morphology figure using medium culture limbal stem cell described in comparative example 3（Form under microscope,
10×）.
Specific embodiment
Present disclosure is further illustrated with reference to Figure of description and specific embodiment, but be 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, that the inventive method, step or condition are made is simple
Modification is replaced, and belongs to the scope of the present invention；If not specializing, technological means used is art technology in embodiment
Conventional meanses known to personnel.
10% I-type collagen solution：1mL I-type collagens are dissolved in 9mL DMEM/F12 culture mediums.
IV clostridiopetidase As：0.2wt% IV collagenase solutions are configured to DMEM/F12 culture mediums.
People recombinates EGF storing solutions：Prepared with PBS, be made into 5 μ g/mL EGF storing solutions.
Insulin stock liquid：Prepared with 0.005 M HCl, concentration is 2.5g/L.
3,3 ', 5-Triiodo-L-Thyronine storing solution：13.6mg 3,3 ', 5-Triiodo-L-Thyronine are molten
Solution adds 85mL PBS in 15 mL 0.02M NaOH；The liquid that 0.1ml is prepared is taken, PBS to 20ml is added, as
Storing solution, concentration is：10-6 M。
Hydrocortisone storing solution：5mg hydrocortisones are dissolved in 1mL absolute ethyl alcohols, add PBS to 25mL, and concentration is
200 µg/mL。
Cholera toxin storing solution：1mg cholera toxins are dissolved in 1mL steril cells culture level pure water, take wherein 0.1mL, plus
Enter PBS to 20mL, concentration is：5ug/mL.
Embodiment 1
Limbal stem cell culture medium：220mL DMEM, 220mL DMEM/F12,50mL FBS, 5mL 100 × dual anti-（100IU
Penicillin, 100ug/ml streptomysins）, 1mL people's restructuring EGF storing solutions, 1mL insulin stock liquid, 1mL 3,3 ', 5-Triiodo-
L-Thyronine storing solutions, 1mL hydrocortisone storing solutions, 1mL cholera toxin storing solutions are positioned over 4 DEG C of preservations, using preceding
37 DEG C of rewarmings.
It is dual anti-with containing in gnotobasis with tissue clamps and corneal scissors clip people's corneal limbal tissue under surgical operation microscope
（Pen .- Strep, 1 ×）PBS rinse 2 times, each 5min；Tissue is shredded with scissors again.
According to the volume of tissue block, per 1cm3Tissue block adds the wt % IV collagen enzyme liquids of 5ml 0.2, and 37 DEG C are softly shaken
After swinging digestion 2h, IV collagen enzyme liquids are removed, the trypsin solutions of 5mL 0.25% are added, after uniform suspension cell, with 100 μm of nets
Filter is sieved through, then is placed in 37 DEG C of further digestion 15min, add the DMEM containing 10%FBS to terminate digestion, 1000rpm centrifugations
5min, reject supernatant.
10% I-type collagen is placed in ice bath, adds the I-type collagens of 1mL 10% to be wrapped per hole in 6 orifice plates
Quilt, is placed in 37 DEG C, containing 5%CO240min is incubated in incubator, is rinsed with PBS, remove PBS.
The resuspended postdigestive cell of limbal stem cell culture medium of 2mL rewarmings is added, is planted respectively in being coated with 10% I
In the hole of collagen type, 37 DEG C are placed in, containing 5%CO2Cultivated in incubator, liquid, and the observation of cell in microscope are changed every other day
Growth conditions.
Embodiment 2
Experimental technique with embodiment 1, it is unique unlike, the limbal stem cell culture medium composition used by the present embodiment is：
204.5mL DMEM, 204.5mL DMEM/F12,75mL FBS, 5mL 100 × dual anti-（100IU penicillin, 100ug/ml strepto-s
Element）, 2mL people's restructuring EGF storing solutions, 2mL insulin stock liquid, 2.5mL 3,3 ', 5-Triiodo-L-Thyronine deposit
Liquid, 2.5mL hydrocortisone storing solutions, 2mL cholera toxin storing solutions.
Comparative example 1
Experimental technique with embodiment 1, it is unique unlike concentration of the EGF in limbal stem cell culture medium be 2ug/ml, its
Result finds that cell growth is slow, loses the form of epidermal cell, and cell is in fusiformis.
Comparative example 2
Experimental technique with embodiment 1, it is unique unlike, serum-concentration is reduced to 2% from 10%（That is the addition of FBS is
10mL）, as a result finding that the cell of cell becomes big, nucleus diminishes, cell attachment ability etc..
Comparative example 3
Experimental technique with embodiment 1, it is unique unlike concentration of the insulin in limbal stem cell culture medium be 2ug/ml,
Its result finds that cell growth is slow, and the form of epidermal cell is not obvious, and cell is loose with Cell tracking, dead cell compared with
It is many.
Claims (5)
1. a kind of limbal stem cell culture medium, it is characterised in that contain following components in culture medium：5mL 100 × dual anti-, 10
~20ng/ml people restructuring EGF, 5~10ug/ml insulin, 1~5x10-9M 3- iodine thyronine, 0.2~1ug/ml
Hydrocortisone, 10~20ng/ml cholera toxins, 10~15% hyclones, balance of DMEM and/or DMEM/F12.
2. a kind of isolated culture method of limbal stem cell, it is characterised in that be corneal edge after corneal limbal tissue is cleaned
Tissue carries out digesting to obtain limbal stem cell, adds the limbal stem cell culture medium described in claim 1 to be cultivated；
The enzymolysis is primary enzymolysis or secondary enzymolysis；The secondary enzymolysis are first to be digested with IV clostridiopetidase As, then use basic protein
Enzyme is digested.
3. the isolated culture method of limbal stem cell according to claim 2, it is characterised in that the IV clostridiopetidase As
Concentration is 0.2~0.3%.
4. the isolated culture method of limbal stem cell according to claim 2, it is characterised in that the IV clostridiopetidase As
Enzymolysis time is 2~3h.
5. the isolated culture method of the limbal stem cell according to claim 2 to 4 any one of people's, it is characterised in that
Before adding limbal stem cell to be cultivated, I-type collagen is first added.
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Cited By (6)
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| CN109321527A (en) * | 2018-10-10 | 2019-02-12 | 中国海洋大学 | The extracorporeal culturing method of limbal stem cell stability |
| CN109439628A (en) * | 2018-10-10 | 2019-03-08 | 中国海洋大学 | Limbal stem cell primary culture method |
| CN109749997A (en) * | 2018-05-11 | 2019-05-14 | 中山大学中山眼科中心 | A kind of Limbus corneae stem cell serum-free culture medium and its cultural method |
| CN112126626A (en) * | 2020-09-30 | 2020-12-25 | 广东康盾生物工程技术有限公司 | 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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| CN109679911A (en) * | 2019-02-26 | 2019-04-26 | 山东大学齐鲁医院 | A kind of limbal stem cell culture medium and cultural method |
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| CN109321527A (en) * | 2018-10-10 | 2019-02-12 | 中国海洋大学 | The extracorporeal culturing method of limbal stem cell stability |
| CN109439628A (en) * | 2018-10-10 | 2019-03-08 | 中国海洋大学 | Limbal stem cell primary culture method |
| CN112126626A (en) * | 2020-09-30 | 2020-12-25 | 广东康盾生物工程技术有限公司 | Limbal stem cell culture medium and culture method |
| 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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