CN105013010B - A kind of laminin film of auxiliary iPS RPE transplanting - Google Patents
A kind of laminin film of auxiliary iPS RPE transplanting Download PDFInfo
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- CN105013010B CN105013010B CN201510398770.0A CN201510398770A CN105013010B CN 105013010 B CN105013010 B CN 105013010B CN 201510398770 A CN201510398770 A CN 201510398770A CN 105013010 B CN105013010 B CN 105013010B
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- laminin
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Abstract
The invention provides a kind of laminin film of auxiliary iPS RPE transplanting and the iPS RPE cell patches as made from the film, the laminin film can promote the retina cell for damaging regression to divide a word with a hyphen at the end of a line, break up, regenerate on the collagen scaffold of offer, itself identical tissue is formed, while disturbs fibroblastic hyperplasia of non-sequence to form scar.The present invention improves the mechanical property of autoserum laminin biomaterial, it can be transplanted in subretinal space, prior art is overcome because mechanical property is weak, immunogenicity is high, it is difficult to degrade and makes the defects of survival rate is low after stem cell transplantation, effectively repair the tissue of lesion, there is provided new treatment method and thinking, reduce the pain of patient.
Description
Technical field
The present invention relates to a kind of protein film, more particularly to a kind of laminin film of auxiliary iPS-RPE transplanting.
Background technology
Intractable PVR is the damage of a kind of retinal tissue or degenerative disease, including age-related macular degeneration,
Stargardt diseases and retinal pigment degeneration etc., illness rate has become me up to more than 20% in more than the 50 years old crowd in China
The main diseases causing blindness that state becomes increasingly conspicuous, its common pathological characters is for retinal pigment epithelium (RPE cells) denaturation and extremely
Die, cause to blind.The development of stem cells technology makes it possible that new RPE cell replacements lack or be denatured RPE cells.
But after cell transplantation, even if applying immunodepressant, transplanted cells also can not long-term surviving, clinical efficacy is not
Significantly, now still can not wide popularization and application.Animal model finds that survival rate is only about after subretinal space is transplanted 7 days
0.01% (finding only have hundreds of cell survivals after the cell transplantation of 200,000 ES cell deriveds).
Transplanted cells are difficult to one of major reason survived：Stem cell is expanded in laboratory culture dish, once move
Planting internal specific site just has propagation difficult.Because new environment is led to full of complicated X factor, the stem cell of transplanting
Often can be dead, or correctly can not be incorporated into perienchyma.Recent studies suggest that hydrogel can promote stem-cell therapy effect,
But its Main Ingredients and Appearance is hyaluronic acid and methylcellulose, the problem of still suffering from heterologous and degrade.Therefore exploitation neoformation material
Material technology, turn into the effective means for improving stem-cell therapy effect.
The content of the invention
A kind of auxiliary iPS-RPE transplanting is provided it is an object of the invention to overcome the shortcomings of the prior art part
Laminin film, as iPS-RPE transplantation carrier, to promote the attaching and survival of cell after transplanting, improve dry thin
Born of the same parents treat the success rate of blinding illness in eye.Present invention also offers the iPS-RPE cell patches as made from the laminin film,
Present invention also offers purposes of the laminin film in cell carrier is prepared.
To achieve the above object, the technical scheme taken：A kind of laminin film of auxiliary iPS-RPE transplanting, institute
State laminin film be after by crosslinking agent, laminin is crosslinked in the solution dry obtained by.
Preferably, crosslinking agent used is glutaraldehyde.The inner pore formation basic theory of spongy collagenous biological diaphragm is equal for its
The ice crystal that liquid in chylema liquid is formed in cross-linking process is freezed determined because serum protein molecule have it is very strong sticky
Property, how high density, the chemistry of high quality collagenous biological film make is in place of thinking and operating difficulties by sticky material
The homogeneous slurry of flowable is formed, how long collagenous fibres are separated out to the fiber for forming high concentration from homogeneous slurry for this
Body is the key of the present invention, and addition glutaraldehyde is unique technology of the invention.
Preferably, concentration of the glutaraldehyde in the solution is 5mg/mL.
Preferably, the crosslinking temperature is 4 DEG C, and the crosslinking time is 24 hours.
Preferably, the drying temperature is 50 DEG C, and drying time is 48 hours.
Preferably, the pore size of the laminin film is 20~80 microns.More preferably 20-60 microns, most preferably
It is worth for 25 microns.
Present invention also offers a kind of iPS-RPE cell patches, the iPS-RPE cell patches are by by iPS-RPE
After cell is inoculated on the laminin film layer Fibronectin film of auxiliary iPS-RPE transplanting described above obtained by culture.
Preferably, before the inoculation, by the laminin film first in the ethanol solution that concentration is 70%~75%
Middle immersion, then soaks in DMEM.In program is freeze-dried, the sterilization of diaphragm, to avoid repopulating cell as far as possible
Afterwards due to diaphragm sterilization not exclusively caused by pollution problem.But diaphragm be autoserum source material, HTHP and often
Rule chemical disinfection method easily causes its degraded.Therefore, the measure that uses of the present invention for：Serum Laminin film is first steeped into ethanol
24hr in solution, then steep the inoculation for being used for cell in DMEM after 24hr.Alcohol molecule has very strong penetration, can pass through thin
The film on bacterium surface, into inside bacterium, make the protein coagulating of composition bacterium Life Base, bacterium is killed.The concentration of alcohol
Difference, purposes also can be otherwise varied.The present invention is used to sterilize using 70%-75% alcohol, because the wine of excessive concentrations
Essence can form layer protecting film in bacterium surface, prevent it from entering in bacterial body, it is difficult to thoroughly to kill bacterium；If alcohol concentration
It is too low, though can then enter bacterium, its internal protein coagulating equally can not thoroughly can not be killed bacterium.Here
The concentration of ethanol solution is volumetric concentration.
Present invention also offers purposes of the laminin film in cell carrier is prepared.Layer adhesion of the present invention
Protein film can be used as stem cell adhesion supporter, prepare iPS-RPE cell patches, the iPS-RPE cell patches of preparation are used
Transplanted in subretinal space, for treating intractable PVR, including age-related macular degeneration, Stargardt diseases and view
Membranochromic pigments denaturation etc..
Laminin diaphragm of the present invention can the degradable absorption in 3-6 months.The iPS-RPE cell patches
The cell concentration that can be applied to any position of subretinal space injection and can survive is high after being injected compared with equal cell suspension
50%.The iPS-RPE cell patches have certain in the degenerative change for preventing or reducing retinal light injury photoreceptor
Purposes.The iPS-RPE cell patches have the purposes for preventing or reducing retinal glial cicatrization.
Laminin film of the present invention can be stored in water, and concentration of the laminin film in water is preferably
1%-4%, more preferably 1.5-3%, optimum value 1.75-2%.Percentage value unit described here is mass/volume.
In a word, the present invention provide it is a kind of using autoserum laminin be material make high concentration, high density, it is micro-/
The preparation method of degradable laminin film without open space.Stem cell is transplanted available for subretinal space, there is provided thin
The impetus that born of the same parents are sticked, cell survival rate is improved, and then treat intractable PVR.
The beneficial effects of the present invention are：The invention provides a kind of auxiliary iPS-RPE transplanting laminin film with
And the iPS-RPE cell patches as made from the film, the laminin film can promote the retina cell for damaging regression to exist
Divide a word with a hyphen at the end of a line, break up, regenerate on the collagen scaffold of offer, form itself identical tissue, while disturb the fibroblastic of non-sequence
Hyperplasia forms scar.The present invention improves the mechanical property of autoserum laminin biomaterial, can be under retina
Chamber is transplanted, and overcomes prior art because mechanical property is weak, and immunogenicity is high, it is difficult to degraded and survival rate is low after making stem cell transplantation
The defects of, effective tissue for repairing lesion, there is provided new treatment method and thinking, reduce the pain of patient.
Brief description of the drawings
Fig. 1 is preparation and the process for using figure of iPS-RPE cell patches of the present invention.
Embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
A kind of embodiment of the preparation method of Laminin ELISA of the present invention, the preparation method are as follows：
Homogenic rat heart takes blood, places 10min, 1000rpm centrifugation 20min, abandons precipitation, stay upper serum, remove
Chylomicron, isolate Serum Laminin；Gel chromatography purifies Serum Laminin, and DEAE cellulose anion displacement chromatographies are pure
Change Serum Laminin and concentrate, obtain the Laminin ELISA.Serum Laminin after concentration is moved into culture dish and stood
In 4 degrees Celsius of refrigerators untill bubble is removed.
A kind of embodiment of the preparation method of the laminin film of auxiliary iPS-RPE transplanting of the present invention, the system
Preparation Method is as follows：
Glutaraldehyde is added into the Laminin ELISA solution of above-mentioned preparation, concentration of the glutaraldehyde in the solution is
5mg/mL, then insert in 4 degrees Celsius of refrigerators and be crosslinked 24hr；It is then placed in after drying 48hr in 50 degrees Celsius of drying bakers and takes out, obtains
To the Laminin ELISA film.The Laminin ELISA film is totally sealed in distilled water flushing standby in refrigerator.
A kind of embodiment of the preparation method of iPS-RPE cell patches of the present invention, the preparation method are as follows：
The Laminin ELISA film of above-mentioned preparation is first steeped into 24hr in the ethanol solution that volumetric concentration is 70%, then steeped into DMEM
It is used for the inoculation of iPS-RPE cells after middle 24hr；IPS-RPE cells are planted on the Laminin ELISA film after above-mentioned processing,
Nutrient solution was changed per 2-3 days.Cell starts to stick laminin film after 2 weeks, is formed on typical cobblestone sample individual layer pigment
Epithelium, serum-free medium is now changed, cell attaches to laminin film completely after 4 weeks, obtains the iPS-RPE cells
Diaphragm.
IPS-RPE cell patches are cut into size and shape needed for transplanting using laser microdissection system, in retina
Cavity of resorption is transplanted, as shown in Figure 1.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention
And scope.
Claims (9)
1. a kind of laminin film of auxiliary iPS-RPE transplanting, it is characterised in that the laminin film is to pass through crosslinking
Obtained by agent is dried after laminin is crosslinked in the solution；
Crosslinking agent used is glutaraldehyde.
2. the laminin film of auxiliary iPS-RPE transplanting according to claim 1, it is characterised in that the glutaraldehyde
Concentration in the solution is 5mg/mL.
3. the laminin film of auxiliary iPS-RPE according to claim 1 transplanting, it is characterised in that the solution is
Water.
4. the laminin film of auxiliary iPS-RPE transplanting according to claim 1, it is characterised in that the crosslinking temperature
Spend for 4 DEG C, the crosslinking time is 24 hours.
5. the laminin film of auxiliary iPS-RPE transplanting according to claim 1, it is characterised in that the dry temperature
Spend for 50 DEG C, drying time is 48 hours.
6. the laminin film of auxiliary iPS-RPE transplanting according to claim 1, it is characterised in that the layer adhesion
The pore size of protein film is 20~80 microns.
7. a kind of iPS-RPE cell patches, it is characterised in that the iPS-RPE cell patches are by the way that iPS-RPE cells are connect
Kind on the laminin film that described auxiliary iPS-RPE as any such as claim 1-5 is transplanted afterwards obtained by culture.
8. iPS-RPE cell patches according to claim 7, it is characterised in that before the inoculation, by the layer adhesion
Protein film first soaks in the ethanol solution that concentration is 70%~75%, is then soaked in DMEM.
9. use of the laminin film of any described auxiliary iPS-RPE transplanting of claim 1-6 in cell carrier is prepared
On the way.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510398770.0A CN105013010B (en) | 2015-07-07 | 2015-07-07 | A kind of laminin film of auxiliary iPS RPE transplanting |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510398770.0A CN105013010B (en) | 2015-07-07 | 2015-07-07 | A kind of laminin film of auxiliary iPS RPE transplanting |
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| CN105013010A CN105013010A (en) | 2015-11-04 |
| CN105013010B true CN105013010B (en) | 2018-01-12 |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105597145A (en) * | 2016-02-22 | 2016-05-25 | 中山大学中山眼科中心 | Retina cell scaffold biological surgical binder and preparing method thereof |
| CN114540302A (en) * | 2022-01-25 | 2022-05-27 | 中山大学中山眼科中心 | Method for generating hiPSC-RPE cell patch by combining biological matrix glue |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100560142C (en) * | 2003-10-10 | 2009-11-18 | 细胞生物工程公司 | Be used on biopolymer, cultivating the method and composition of endothelial cell and relevant cell and generation artifical corneal transplants |
| GB0806746D0 (en) * | 2008-04-14 | 2008-05-14 | Ucl Business Plc | Membrane |
| CN101380486B (en) * | 2008-10-28 | 2012-05-30 | 暨南大学 | Active regeneration artificial cornea graft and preparation method thereof |
| DK2780022T3 (en) * | 2011-11-14 | 2019-07-15 | Astellas Inst For Regenerative Medicine | PHARMACEUTICAL PREPARATIONS OF HUMAN RPE CELLS AND USES THEREOF |
| CN103087533B (en) * | 2013-01-17 | 2015-11-25 | 黑龙江省重生生物科技有限公司 | A kind of Biological gel membrane for cell cultures, preparation method and application thereof |
| JP6518879B2 (en) * | 2013-10-09 | 2019-05-29 | 株式会社ヘリオス | Method for purification of retinal pigment epithelial cells |
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