CN104178452A - Retina pigment epithelial cell culture medium and application thereof - Google Patents
Retina pigment epithelial cell culture medium and application thereof Download PDFInfo
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- CN104178452A CN104178452A CN201410424794.4A CN201410424794A CN104178452A CN 104178452 A CN104178452 A CN 104178452A CN 201410424794 A CN201410424794 A CN 201410424794A CN 104178452 A CN104178452 A CN 104178452A
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Abstract
The invention relates to a retina pigment epithelial cell culture medium and an application thereof. The retina pigment epithelial cell culture medium comprises a first RPE (Retinal Pigment Epithelium) cell culture medium and a second RPE cell culture medium, wherein the first RPE cell culture medium and the second RPE cell culture medium are respectively used in 1.5 weeks, a RPE monolayer cell needed for cell replacement therapy can be quickly cultured; and the cultured RPE is polygonal, embedded and polarly-arranged monolayer cell and has the normal biological functions. Compared with the prior art, the RPE monolayer cell needed for cell replacement therapy can be quickly cultured in 1.5 weeks under the precondition that the conventional laboratory consumables are used; and the retina pigment epithelial cell culture medium is relatively low in cost and high in proliferation speed.
Description
Technical field
The present invention relates to a kind of substratum and uses thereof, especially relate to a kind of retinal pigment epithelium substratum and application thereof, belong to biomedical sector.
Background technology
Senile macular degeneration SMD (Age-related Macular Degeneration, AMD) is a kind of fundus oculi disease multiple in elderly population.Centered by AMD clinical manifestation, carrying out property of eyesight goes down, thereby causes reading and other behavior disorders.The formation of previous research work discovery AMD is engulfed digestion ability to retinal pigment epithelium (RPE) to acromere dish film and is declined relevant, not by the dish film residual body retention of complete digestion in basis pontis cell magma, and discharge and be deposited on Bruch film to extracellular, form glassy membrane wart.Except cell replacement therapy, there is no at present the effective ways that reverse this degeneration process and recover eyesight.Cell replacement therapy is exactly the RPE cell of peeling off patient's pathology by surgical operation, the subretinal space of again the RPE cell in various sources being transplanted to RPE hereditary defect animal taking suspension or polymer support as carrier, can effectively delay the degeneration of photoreceptor cell and improve sight function.
RPE is the continuous monolayer cell between Choroid and neural retina.Its function is mainly the self that maintains retina cell, and this comprises engulfs the acromere dish film that digests the photoreceptor cell coming off; Promote to look the regeneration of medium 11-cis retinal important in circulation; Regulate the immune response of intraocular; Participate in forming retina-blood vessel barrier etc.And this physiological function of RPE cell generation abnormal and AMD is closely related.In cell replacement therapy, the donor source that at present conventional RPE transplants is mainly fetus and grownup's RPE.Although induced multi-potent stem cells (iPSC) does not relate to ethnics Problem such as destroying embryonic cell, and has avoided immunological rejection, the possible tumorigenicity of iPSC has limited its application in treatment AMD.
At present, be used for transplanting the fetus of RPE and grownup's RPE quantity is very limited, can not meet patient's demand.Meanwhile, under RPE culture condition in the past, not only need to use the expensive consumptive materials such as transwell, and RPE amplification rate is slow, can not form fast the RPE monolayer with polarity and biological function.These have all seriously fettered the application of RPE cell replacement therapy.
Summary of the invention
Object of the present invention is exactly to provide a kind of retinal pigment epithelium substratum and application thereof in order to overcome the defect that above-mentioned prior art exists.
Object of the present invention can be achieved through the following technical solutions:
A kind of retinal pigment epithelium substratum, comprises a RPE cell culture medium and the 2nd RPE cell culture medium;
A described RPE cell culture medium comprises following component, in 500ml, and each component as follows with magnitude relation:
α modifies MEM: 380-405ml;
N1 fill-in: 5ml;
Glutamine: 5mL:
Penicillin-Streptomycin sulphate; 5mL;
Non-essential amino acid: 5mL:
Taurine: 100-150mg;
Hydrocortisone: 10-15 μ g;
Trilute: 0.008-0.012 μ g;
Heat-inactivated fetal bovine serum: 75-100ml;
The 2nd described RPE cell culture medium comprises following component, in 500ml, and each component as follows with magnitude relation:
α modifies MEM: 430-455ml;
N1 fill-in: 5ml;
Glutamine: 5mL;
Penicillin-Streptomycin sulphate: 5mL;
Non-essential amino acid: 5mL;
Taurine: 75-100mg;
Hydrocortisone: 5-10 μ g;
Trilute: 0.003-0.008 μ g;
Heat-inactivated fetal bovine serum: 25-50ml.
A described RPE cell culture medium or each component of the 2nd RPE cell culture medium all by the filter media in 0.22 μ M aperture, are used for removing the polluted bacteria that may exist by the filter media in 0.22 μ M aperture before preparation substratum.An above-mentioned RPE cell culture medium or the 2nd RPE cell culture medium can be stablized preservation 3 months at 4 DEG C.
Retinal pigment epithelium substratum is for the amplification of Embryonic Retina pigment epithelial cell rapid in-vitro.Specifically comprise the following steps:
(1) 1～6 hour in advance, modify MEM with α and be coated with culturing bottle, wherein the ln concentration of α modification MEM is 5～20 μ g/mL;
(2) the embryo RPE cell that thaws in 36～38 DEG C of water baths fast, takes out cryopreservation tube;
(3) the embryo RPE in cryopreservation tube is transferred in taper test tube, and add a RPE cell culture medium, centrifugal, centrifugal operational condition is normal temperature, and rotating speed is 300～500x g, and centrifugation time is 3～5 minutes;
(4) supernatant in step (3) taper test tube is siphoned away, and then add a RPE cell culture medium re-suspended cell;
(5) substratum of culturing bottle in step (1) is siphoned away, the cell after simultaneously adding step (4) resuspended, mixes;
(6) culturing bottle is positioned in incubator and is cultivated, the condition of incubator is 36～38 DEG C, 4.5～5.5%CO
2, 90～100% humidity, change the substratum in culturing bottle into the 2nd RPE cell culture medium on the 3rd day;
(7) after, within every 3 days, change the second RPE cell culture medium one time, after 1.5 weeks, can be observed the monolayer cell that there is melanic polygon, inlays, is polarity arrangement.Cell number in step (7) after amplification is greater than 500,000,000.
At present, be used for transplanting the fetus of RPE and grownup's RPE quantity is very limited, can not meet patient's demand.Meanwhile, under RPE culture condition in the past, not only need to use the expensive consumptive materials such as transwell, and RPE amplification rate is slow, can not forms fast the RPE monolayer with polarity and biological function.The invention provides a kind of culture medium prescription of Embryonic Retina pigment epithelial cell rapid in-vitro amplification, using under the prerequisite of normal experiment consumptive material, in 1.5 weeks, can fast culture go out the required RPE monolayer cell of cell replacement therapy.
Compared with prior art, the invention has the advantages that:
1, in substratum of the present invention, the proportioning of each component is selected to ensure the optimal growth of retinal pigment epithelium, if adopt other proportioning all can not reach the optimal growth of retinal pigment epithelium.Simultaneously in substratum, add taurine, hydrocortisone and trilute, and adopt proportioning of the present invention can simulate the retinal pigment epithelium life condition under environment prevent the oxidative damage of retinal pigment epithelium in vivo.
2, the present invention adopts a RPE cell culture medium and the component concentration of the 2nd RPE cell culture medium change, and a RPE cell culture medium, by the increase amount of serum RPE cell that increases rapidly, is avoided the too low cellular form generation irreversible change that causes of cell concentration.The corresponding consumption that increases taurine, hydrocortisone and trilute of component of the 2nd RPE cell culture medium is mainly the vigor that strengthens RPE cell; Reduce amount of serum simultaneously and can avoid cell hyperproliferation and apoptosis, and maintain monolayer cell, prevent that the irregular agglomerate of multi-layer cellular from occurring.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
The required reagent source of the present embodiment is as follows:
A substratum for Embryonic Retina pigment epithelial cell rapid in-vitro amplification, is made up of a RPE cell culture medium and the 2nd RPE cell culture medium respectively.A described RPE cell culture medium or each component of the 2nd RPE cell culture medium all by the filter media in 0.22 μ M aperture, are used for removing the polluted bacteria that may exist by the filter media in 0.22 μ M aperture before preparation substratum.Its formula and preparation method are as follows:
1) a RPE cell culture medium (filtering by 0.22 μ M):
2) the 2nd RPE cell culture medium (filtering by 0.22 μ M):
Should mix at normal temperatures in mentioned reagent, filter by 0.22 μ M.Above-mentioned substratum can be stablized preservation 3 months at 4 DEG C.
A purposes for above-mentioned substratum, comprises following steps:
(1) 2 hours in advance, the α that contains 20 μ g lns with 2mL modified the coated T25 culturing bottle of MEM;
(2) the embryo RPE cell that thaws in 37 DEG C of water baths fast, takes out cryopreservation tube;
(3) the embryo RPE in cryopreservation tube is transferred in 1 15mL taper test tube, and add 5mL the one RPE cell culture medium, centrifugal 5 minutes of 300x g;
(4) supernatant in step (3) taper test tube is siphoned away, then add 5mL the one RPE cell culture medium re-suspended cell;
(5) substratum of T25 culturing bottle in step (1) is siphoned away, the cell after simultaneously adding step (4) resuspended, mixes gently;
(6) be positioned in incubator and cultivate, within the 3rd day, use 5mL the 2nd RPE cell culture medium instead;
(7) within every 3 days, change the second RPE cell culture medium one time, after 1.5 weeks, can be observed the monolayer cell that there is melanic polygon, inlays, is polarity arrangement.
Embodiment 2
A kind of retinal pigment epithelium substratum, comprises a RPE cell culture medium and the 2nd RPE cell culture medium:
The one RPE cell culture medium comprises following component, in 500ml, and each component as follows with magnitude relation:
α modifies MEM: 380ml, N1 fill-in: 5ml, glutamine: 5mL, penicillin-Streptomycin sulphate: 5mL, non-essential amino acid: 5mL, taurine: 100mg, hydrocortisone: 10 μ g, trilute: 0.008 μ g, heat-inactivated fetal bovine serum: 100ml;
The 2nd RPE cell culture medium comprises following component, in 500ml, and each component as follows with magnitude relation:
α modifies MEM: 430ml, N1 fill-in: 5ml, glutamine: 5mL, penicillin-Streptomycin sulphate: 5mL, non-essential amino acid: 5mL, taurine: 75mg, hydrocortisone: 5 μ g, trilute: 0.003 μ g, heat-inactivated fetal bovine serum: 50ml.
Each component of the one RPE cell culture medium or the 2nd RPE cell culture medium all by the filter media in 0.22 μ M aperture, is used for removing the polluted bacteria that may exist by the filter media in 0.22 μ M aperture before preparation substratum.An above-mentioned RPE cell culture medium or the 2nd RPE cell culture medium can be stablized preservation 3 months at 4 DEG C.
Retinal pigment epithelium substratum is for the amplification of Embryonic Retina pigment epithelial cell rapid in-vitro.Specifically comprise the following steps:
(1) 1 hour in advance, modify MEM with α and be coated with culturing bottle, wherein the ln concentration of α modification MEM is 5 μ g/mL;
(2) the embryo RPE cell that thaws in 36 DEG C of water baths fast, takes out cryopreservation tube;
(3) the embryo RPE in cryopreservation tube is transferred in taper test tube, and add a RPE cell culture medium, centrifugal, centrifugal operational condition is normal temperature, and rotating speed is 300x g, and centrifugation time is 5 minutes;
(4) supernatant in step (3) taper test tube is siphoned away, and then add a RPE cell culture medium re-suspended cell;
(5) substratum of culturing bottle in step (1) is siphoned away, the cell after simultaneously adding step (4) resuspended, mixes:
(6) culturing bottle is positioned in incubator and is cultivated, the condition of incubator is 36 DEG C, 4.5%CO
2, 90% humidity, changes the substratum in culturing bottle into the 2nd RPE cell culture medium on the 3rd day:
(7) after, within every 3 days, change the second RPE cell culture medium one time, after 1.5 weeks, can be observed the monolayer cell that there is melanic polygon, inlays, is polarity arrangement.Cell number in step (7) after amplification is greater than 500,000,000.
Embodiment 3
A kind of retinal pigment epithelium substratum, comprises a RPE cell culture medium and the 2nd RPE cell culture medium;
The one RPE cell culture medium comprises following component, in 500ml, and each component as follows with magnitude relation:
α modifies MEM: 405ml, N1 fill-in: 5ml, glutamine: 5mL, penicillin-Streptomycin sulphate: 5mL, non-essential amino acid: 5mL, taurine: 150mg, hydrocortisone: 15 μ g, trilute: 0.012 μ g, heat-inactivated fetal bovine serum: 75ml;
The 2nd RPE cell culture medium comprises following component, in 500ml, and each component as follows with magnitude relation:
α modifies MEM: 455ml, N1 fill-in: 5ml, glutamine: 5mL, penicillin-Streptomycin sulphate: 5mL, non-essential amino acid: 5mL, taurine: 100mg, hydrocortisone: 10 μ g, trilute: 0.008 μ g, heat-inactivated fetal bovine serum: 25ml.
Each component of the one RPE cell culture medium or the 2nd RPE cell culture medium all by the filter media in 0.22 μ M aperture, is used for removing the polluted bacteria that may exist by the filter media in 0.22 μ M aperture before preparation substratum.An above-mentioned RPE cell culture medium or the 2nd RPE cell culture medium can be stablized preservation 3 months at 4 DEG C.
Retinal pigment epithelium substratum is for the amplification of Embryonic Retina pigment epithelial cell rapid in-vitro.Specifically comprise the following steps:
(1) 6 hours in advance, modify MEM with α and be coated with culturing bottle, wherein the ln concentration of α modification MEM is 20 μ g/mL;
(2) the embryo RPE cell that thaws in 38 DEG C of water baths fast, takes out cryopreservation tube;
(3) the embryo RPE in cryopreservation tube is transferred in taper test tube, and add a RPE cell culture medium, centrifugal, centrifugal operational condition is normal temperature, and rotating speed is 500x g, and centrifugation time is 3 minutes;
(4) supernatant in step (3) taper test tube is siphoned away, and then add a RPE cell culture medium re-suspended cell;
(5) substratum of culturing bottle in step (1) is siphoned away, the cell after simultaneously adding step (4) resuspended, mixes;
(6) culturing bottle is positioned in incubator and is cultivated, the condition of incubator is 38 DEG C, 5.5%CO
2, 100% humidity, changes the substratum in culturing bottle into the 2nd RPE cell culture medium on the 3rd day;
(7) after, within every 3 days, change the second RPE cell culture medium one time, after 1.5 weeks, can be observed the monolayer cell that there is melanic polygon, inlays, is polarity arrangement.Cell number in step (7) after amplification is greater than 500,000,000.
Embodiment 4
A kind of retinal pigment epithelium substratum, comprises a RPE cell culture medium and the 2nd RPE cell culture medium;
The one RPE cell culture medium comprises following component, in 500ml, and each component as follows with magnitude relation:
α modifies MEM: 390ml, N1 fill-in: 5ml, glutamine: 5mL, penicillin-Streptomycin sulphate: 5mL, non-essential amino acid: 5mL, taurine: 120mg, hydrocortisone: 13 μ g, trilute: 0.010 μ g, heat-inactivated fetal bovine serum: 90ml;
The 2nd RPE cell culture medium comprises following component, in 500ml, and each component as follows with magnitude relation:
α modifies MEM: 440ml, N1 fill-in: 5ml, glutamine: 5mL, penicillin-Streptomycin sulphate: 5mL, non-essential amino acid: 5mL, taurine: 90mg, hydrocortisone: 8 μ g, trilute: 0.005 μ g, heat-inactivated fetal bovine serum: 40ml.
Each component of the one RPE cell culture medium or the 2nd RPE cell culture medium all by the filter media in 0.22 μ M aperture, is used for removing the polluted bacteria that may exist by the filter media in 0.22 μ M aperture before preparation substratum.An above-mentioned RPE cell culture medium or the 2nd RPE cell culture medium can be stablized preservation 3 months at 4 DEG C.
Retinal pigment epithelium substratum is for the amplification of Embryonic Retina pigment epithelial cell rapid in-vitro.Specifically comprise the following steps:
(1) 3 hours in advance, modify MEM with α and be coated with culturing bottle, wherein the ln concentration of α modification MEM is 10 μ g/mL;
(2) the embryo RPE cell that thaws in 37 DEG C of water baths fast, takes out cryopreservation tube;
(3) the embryo RPE in cryopreservation tube is transferred in taper test tube, and add a RPE cell culture medium, centrifugal, centrifugal operational condition is normal temperature, and rotating speed is 400x g, and centrifugation time is 4 minutes;
(4) supernatant in step (3) taper test tube is siphoned away, and then add a RPE cell culture medium re-suspended cell;
(5) substratum of culturing bottle in step (1) is siphoned away, the cell after simultaneously adding step (4) resuspended, mixes;
(6) culturing bottle is positioned in incubator and is cultivated, the condition of incubator is 37 DEG C, 5.0%CO
2, 95.0% humidity, changes the substratum in culturing bottle into the 2nd RPE cell culture medium on the 3rd day;
(7) after, within every 3 days, change the second RPE cell culture medium one time, after 1.5 weeks, can be observed the monolayer cell that there is melanic polygon, inlays, is polarity arrangement.Cell number in step (7) after amplification is greater than 500,000,000.
The above-mentioned description to embodiment is can understand and use invention for ease of those skilled in the art.Person skilled in the art obviously can easily make various amendments to these embodiment, and General Principle described herein is applied in other embodiment and needn't passes through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, those skilled in the art are according to announcement of the present invention, and not departing from improvement and the amendment that category of the present invention makes all should be within protection scope of the present invention.
Claims (7)
1. a retinal pigment epithelium substratum, is characterized in that, comprises a RPE cell culture medium and the 2nd RPE cell culture medium;
A described RPE cell culture medium comprises following component, in 500ml, and each component as follows with magnitude relation:
α modifies MEM: 380-405ml;
N1 fill-in: 5ml;
Glutamine: 5mL;
Penicillin-Streptomycin sulphate: 5mL;
Non-essential amino acid: 5mL;
Taurine: 100-150mg;
Hydrocortisone: 10-15 μ g;
Trilute: 0.008-0.012 μ g;
Heat-inactivated fetal bovine serum: 75-100ml;
The 2nd described RPE cell culture medium comprises following component, in 500ml, and each component as follows with magnitude relation:
α modifies MEM: 430-455ml;
N1 fill-in: 5ml;
Glutamine: 5mL;
Penicillin-Streptomycin sulphate: 5mL;
Non-essential amino acid: 5mL;
Taurine: 75-100mg;
Hydrocortisone: 5-10 μ g;
Trilute: 0.003-0.008 μ g;
Heat-inactivated fetal bovine serum: 25-50ml.
2. a kind of retinal pigment epithelium substratum according to claim 1, it is characterized in that, a described RPE cell culture medium or each component of the 2nd RPE cell culture medium all by the filter media in 0.22 μ M aperture, are removed the miscellaneous bacteria that may exist before preparation substratum.
3. an application for retinal pigment epithelium substratum as claimed in claim 1, is characterized in that, retinal pigment epithelium substratum is for the amplification of Embryonic Retina pigment epithelial cell rapid in-vitro.
4. the application of a kind of retinal pigment epithelium substratum according to claim 3, is characterized in that, retinal pigment epithelium substratum, for the amplification of Embryonic Retina pigment epithelial cell rapid in-vitro, specifically comprises the following steps:
(1) 1～6 hour in advance, modify MEM with α and be coated with culturing bottle;
(2) the embryo RPE cell that thaws in 36～38 DEG C of water baths fast, takes out cryopreservation tube;
(3) the embryo RPE in cryopreservation tube is transferred in taper test tube, and add a RPE cell culture medium, centrifugal;
(4) supernatant in step (3) taper test tube is siphoned away, and then add a RPE cell culture medium re-suspended cell;
(5) substratum of culturing bottle in step (1) is siphoned away, the cell after simultaneously adding step (4) resuspended, mixes;
(6) culturing bottle is positioned in incubator and is cultivated, within the 3rd day, change the substratum in culturing bottle into the 2nd RPE cell culture medium;
(7) after, within every 3 days, change the second RPE cell culture medium one time, after 1.5 weeks, can be observed the monolayer cell that there is melanic polygon, inlays, is polarity arrangement.
5. the substratum of a kind of Embryonic Retina pigment epithelial cell rapid in-vitro amplification according to claim 4, is characterized in that, the ln concentration that the described α of step (1) modifies MEM is 5～20 μ g/mL.
6. the substratum of a kind of Embryonic Retina pigment epithelial cell rapid in-vitro amplification according to claim 4, is characterized in that, in step (3), centrifugal operational condition is normal temperature, and rotating speed is 300～500xg, and centrifugation time is 3～5 minutes.
7. the substratum of a kind of Embryonic Retina pigment epithelial cell rapid in-vitro amplification according to claim 4, is characterized in that, in step (6), the condition of incubator is 36～38 DEG C, 4.5～5.5%CO
2, 90～100% humidity.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106282096A (en) * | 2016-10-19 | 2017-01-04 | 江苏艾尔康生物医药科技有限公司 | A kind of isolated culture method of human retinal pigment epithelial cells layer |
| CN107304412A (en) * | 2016-04-22 | 2017-10-31 | 南京医科大学第附属医院 | The culture medium of retinal pigment epithelium and its application |
| CN107787960A (en) * | 2016-09-07 | 2018-03-13 | 南京医科大学第附属医院 | The frozen stock solution of retinal pigment epithelium and its application |
| CN110106147A (en) * | 2018-04-18 | 2019-08-09 | 浙江大学 | A kind of method and its application that induction human amnion membrane breaks up to retinal photoreceptor cells |
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| CN102465111A (en) * | 2010-11-19 | 2012-05-23 | 薛志刚 | Test method for induced differentiating human embryo stem cells to retina pigment epithelial cells |
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| CN102465111A (en) * | 2010-11-19 | 2012-05-23 | 薛志刚 | Test method for induced differentiating human embryo stem cells to retina pigment epithelial cells |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107304412A (en) * | 2016-04-22 | 2017-10-31 | 南京医科大学第附属医院 | The culture medium of retinal pigment epithelium and its application |
| CN107787960A (en) * | 2016-09-07 | 2018-03-13 | 南京医科大学第附属医院 | The frozen stock solution of retinal pigment epithelium and its application |
| CN107787960B (en) * | 2016-09-07 | 2021-04-02 | 南京医科大学第一附属医院 | Cryopreservation liquid for retinal pigment epithelial cells and application thereof |
| CN106282096A (en) * | 2016-10-19 | 2017-01-04 | 江苏艾尔康生物医药科技有限公司 | A kind of isolated culture method of human retinal pigment epithelial cells layer |
| CN110106147A (en) * | 2018-04-18 | 2019-08-09 | 浙江大学 | A kind of method and its application that induction human amnion membrane breaks up to retinal photoreceptor cells |
| CN110106147B (en) * | 2018-04-18 | 2021-04-13 | 浙江大学 | Method for inducing differentiation of human amniotic epithelial cells into retinal photoreceptor cells and application thereof |
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