CN109868257A - A kind of digestion dissociating method of retinal pigment epithelium primary tissue - Google Patents
A kind of digestion dissociating method of retinal pigment epithelium primary tissue Download PDFInfo
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- CN109868257A CN109868257A CN201810694163.2A CN201810694163A CN109868257A CN 109868257 A CN109868257 A CN 109868257A CN 201810694163 A CN201810694163 A CN 201810694163A CN 109868257 A CN109868257 A CN 109868257A
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- retinal pigment
- pigment epithelium
- eyecup
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Abstract
The invention discloses a kind of digestion dissociating methods of retinal pigment epithelium primary tissue, it is the following steps are included: layer of retina overturning in eyecup is put it into digestive ferment again later towards outside, it is reduced to influence of the digestive ferment to retinal pigment epithelium minimum, obtains more retinal pigment epitheliums while ensureing retinal pigment epithelium purity.This method method has wide application prospect.
Description
Technical field
Present invention relates in general to cell engineering fields, and in particular to a kind of primary group of retinal pigment epithelium
The digestion dissociating method knitted.
Background technique
RPE (source of people retinal pigment epithelium) is the continuous cell monolayer outside retina, because of its sieve-like
Form and cytoplasm in melanin abundant and gain the name, they play important in the maintenance and self-renewing of retina cell
Function, such as phagocytosis digest the acromere (OS) of the photoreceptor cell to fall off, promote the cis- view of medium 11- important in view circulation
The regeneration of yellow aldehyde adjusts intraocular immune response, participates in forming retina-vascular barrier etc..This physiological function of RPE cell
Exception be considered closely related to the generation of related ophthalmology disease, such as RPE barrier cell dysfunction then easily leads to Best
Vitelliform macular malnutrition (Bestvitelliform macular dystrophy, BVMD) and adult vitelliform macular battalion
Support bad (adult-onset vitelliformmacular dystrophy, AVMD).RPE basilar memebrane and Bruch ' s
Connection or dysfunction between membrane are considered age-related property macular degeneration (age-relatedmacular
Degeneration, AMD) it is related to the generation of Sorsby ' s fundus dystrophy.
Currently there are retinal pigment epithelium primary tissue isolation technics mainly by enzymic digestion, institute's used time
Between it is general longer, and the cell quantity separated is on the low side, and the efficiency separated between every batch of is different, and culture effect is bad, after culture
Cell yield is different, thus the peculiar functional expression loss of source of people retinal pigment epithelium is larger.
Summary of the invention
The present invention overcomes the above-mentioned technological deficiency of the prior art, and providing one kind can under the premise of not influencing cell function
Increase substantially the new side of removing retinal pigment epithelium (RPE) primary tissue of the stable productivity of primary cell pick-up rate
Method, the present invention will provide reason to the research of retinal pigment epithelium (preferably to source of people class retinal pigment epithelium)
By support, provide convenience to the passage, preparation, research of cell.
The present invention provides a kind of digestion dissociating method/removing sides of retinal pigment epithelium (RPE) primary tissue
Eyecup is put into digestive ferment by method by the method for the present invention, and compared with the prior art, interior at the same time, digestive ferment is to view
The influence of membranochromic pigments epithelial cell is reduced to minimum, the more views of acquisition while ensureing retinal pigment epithelium purity
Retinal pigment epithelial cell.
The digestion dissociating method of retinal pigment epithelium (RPE) primary tissue proposed by the present invention comprising step:
Eyecup is trimmed, the integrality of eyecup is kept, eyecup is overturn, the inside and outside anti-cap sequence overturn is formed, makes on retinal pigment
Skin cell layer is overturn towards outside, is then placed into separation enzyme solutions and is digested, obtains the retinal pigment epithelium.
The digestion dissociating method of retinal pigment epithelium (RPE) primary tissue proposed by the present invention comprising following
Step:
(1) in vitro eyeball tissue outer surface excess tissue is removed, cuts off ocular tissue from cornea 2mm；
(2) eyecup is trimmed, ensures eyecup integrality；
(3) eyecup is gently squeezed, will be overturn inside and outside layer of retina in eyecup, non-RPE cellular layer turns up in inside, formation
The anti-cap sequence turned；
(4) eyecup after overturning is put into separation enzyme solutions, retinal pigment epithelium layer comes into full contact with separation enzyme
Solution is digested；
(5) after digesting, then with angle tweezer retinal pigment epithelium layer is removed manually；
(6) retinal pigment epithelium of removing is transferred in culture bottle and is cultivated, obtain the retinal color
Plain epithelial cell.
Wherein, the retinal pigment epithelium is the source of people retinal pigment epithelium in eyeball tissue source.
Wherein, the retinal pigment epithelium is the source of people retinal pigment epithelium in primary source.
In step (2), the trimming eyecup are as follows: cut off ocular tissue along cornea 2mm, cut off along ocular tissue's outer diameter, removed
Vitreum, neural confluent monolayer cells, and the tissues such as cornea, iris, crystalline lens are removed.
Step (3) treatment process carries out in PBS.
In step (4), the separation enzyme is II solution of Dispase.
In step (4), it is described separation enzyme the preparation method comprises the following steps: by Dispase II with DPBS solution dissolve, be configured to 2-
The solution of 40mg/ml concentration.
In step (4), the temperature of the digestion is 35 DEG C~39 DEG C；It preferably, is 37 DEG C.
In step (4), the time of the digestion is 10min~65min；It preferably, is 20min~50min.
In step (6), the culture bottle is cell culture T25 culture bottle.
In step (6), the inoculum concentration of the retinal pigment epithelium is cell/bottle of eyeball tissue removing.
In step (6), the method for the culture are as follows: in T25 culture bottle, using RPE culture medium, cultivate the retina
Pigment epithelial cell changes liquid 1 time in 2 days or 3 days, each 5ml.
In step (6), the temperature of the culture is 35 DEG C~39 DEG C；It preferably, is 37 DEG C.
In step (6), the time of the culture is 230-250 hours.Preferably, the time of the culture is 240 hours.
After cultivating the retinal pigment epithelium transfer of removing, the method also includes step (7): by step
(6) the step of retinal pigment epithelium obtained after cultivating is digested, is counted, being frozen.
In step (7), the method for the digestion are as follows: suck culture medium, Tryple select 2ml is added, is put into dioxy
Change and is digested in carbon incubator.
In step (7), the temperature of the digestion is 35 DEG C~39 DEG C；It preferably, is 37 DEG C.
In step (7), the time of the digestion is 8min~12min；It preferably, is 10min.
The present invention also proposes a kind of primary group of retinal pigment epithelium being prepared by above-mentioned digestion dissociating method
It knits.
Wherein, the retinal pigment epithelium is Human RPE Cells in Vitro.
Use digestion dissociating method of the invention, comprising: trimming eyecup removes vitreum, removes neural confluent monolayer cells, ensures
Eyecup integrality；Eyecup is gently squeezed, will be overturn inside and outside retinal pigment epithelium layer, choroid layer is in inside, formation
The anti-cap sequence of outer overturning；I.e. before overturning, due to not cutting off eyecup, the integrality of eyecup is kept, and improve digestive ferment
Concentration shortens digestion time.Therefore, (1) present invention can be while increasing digestion power of the digestive ferment to eyecup, and reduction disappears
Change digestion of the enzyme to non-RPE cell, and then reduces non-RPE cell contamination.(2) since the method for the present invention is without destroying entire eyecup
Structure, keep the integrality of eyecup, make subsequent mechanical separation obtain the biggish complete RPE confluent monolayer cells of area so that obtain
While retinal pigment epithelium purity improves, primary cell amount is also improved.(3) since non-RPE cell contamination subtracts
Few, cell uniformity improves, and purity rises, culture effect enhancing.(4) present invention is without being cut into similar petal-shaped, energy for eyecup
Enough avoid divided area not of uniform size, separation enzyme and cell contact area is inconsistent, efficiency is inconsistent problem.
Beneficial effect of the present invention includes, and compared with digestion process already present on existing market, the present invention, which uses, to disappear
Change distancing method tool purity, in terms of be obviously improved.Compared with the prior art, at the same time, this
Digestive ferment in inventive method is more abundant to the digestion of RPE cell, and the digestion of non-RPE cell is reduced to it is minimum, rear
Continuous stripping bench reduces the influence of non-RPE cell, saves the integrality of RPE cellular layer to the maximum extent, is ensureing RPE cell
More retinal pigment epitheliums are obtained while purity.
Detailed description of the invention
Fig. 1 shows eyecup schematic diagrames after normal eyecup and overturning.
Fig. 2 indicates to digest schematic diagram using the source of people retinal pigment epithelium of the present invention and conventional method, in Fig. 2,
Upper figure expression is in petal-like eyecup after trimming according to existing method；After following figure expression is trimmed according to mode provided by the invention
Eyecup.
Fig. 3 indicates the source of people retinal pigment epithelium quantity schematic diagram using present invention digestion dissociating method removing.
Fig. 4 indicates to illustrate using the source of people retinal pigment epithelium culture amount of present invention digestion dissociating method removing
Figure.
Fig. 5 indicates the source of people retinal pigment epithelium quantity schematic diagram of traditional degrading process removing that disappears.
Fig. 6 indicates the source of people retinal pigment epithelium culture amount schematic diagram using tradition digestion dissociating method removing.
Specific embodiment
In conjunction with following specific embodiments and attached drawing, the present invention is described in further detail.Implement process of the invention,
Condition, experimental method etc. are among the general principles and common general knowledge in the art, this hair in addition to what is specifically mentioned below
It is bright that there are no special restrictions to content.It provides embodiment to be for illustration purposes only, should not be construed as limiting this hair
Bright range or content.
Glutamine, nonessential amino acid in following embodiment are purchased from LIFE TECHOLOGISE company；DPBS(CTS)
Purchased from GIBCO company；Dispase II is purchased from ROCHE Roche Holding Ag；Tryple select is purchased from INVITROGEN company；
α MEM culture medium, N1 additive, taurine, hydrocortisone, trilute are purchased from SIGMA company；
Human blood platelets lysate is purchased from HELIOS company.
Following example 1, embodiment 2 respectively have chosen the source of people view using present invention digestion dissociating method removing
Membranochromic pigments epithelial cell and the source of people retinal pigment epithelium removed using tradition digestion dissociating method.Below two
Culture simultaneously digests embodiment to after being proliferated plateau respectively, number of computations, motility rate.
Embodiment 1: source of people retinal pigment epithelium (RPE) of the invention digests dissociating method
According to the recipe configuration RPE culture medium in table 1.
Table 1: RPE culture medium used in the present invention
| Component | Content |
| α modifies lower bound minimal medium (MEM, α modification) | 500mL |
| N1 additive (N1supplement) | 5mL |
| Glutamine (Glutamine) | 5mL |
| Nonessential amino acid (Non-essentialaminoacids) | 5mL |
| Taurine (Taurine) | 150mg |
| Hydrocortisone (Hydrocortisone) | 15μg |
| Trilute (Triiodo-thyronin) | 0.010μg |
| Human blood platelets lysate (HPL) | 50mL |
Source of people retinal pigment epithelium primary tissue is dissociated in digestion according to the following steps:
(1) sealed membrane for the 15ml centrifuge tube for filling ocular tissue is opened in Biohazard Safety Equipment, it will with 1ml pipettor
Ocular tissue is sucked out, and is put into two holes of the DPBS (CTS) for 12 orifice plates being ready to complete and cleans, cleans 3-5s every time.
(2) ocular tissue is put into the 100mm culture dish for having added about 10ml DPBS (CTS), under Stereo microscope, trimming
Excess surface tissue is removed, the ocular tissue after trimming is moved into the 100mm culture dish that another fills about 10ml DPBS (CTS).
(3) ocular tissue is cut off everywhere along cornea 2mm, remove cornea and prosthomere, keep the integrality of eyecup, use angle tweezer
Eyecup layer of retina is gently squeezed downward, it is seen that white nervous layer gradually falls off, and after nervous layer is completely fallen off, uses angle tweezer
Eyecup is turned, choroid layer is internal, and RPE layers externally, forms the inside and outside anti-cap sequence overturn, is put into 12 orifice plates
It separates in enzyme solutions, is placed in 37.0 DEG C of carbon dioxide incubator and digests, digestion time is 20 ± 5min.
(4) after digesting, eyecup is put into the 100mm culture dish that the 3rd fills about 10ml DPBS (CTS), body formula
Under microscope, with two tissue clamps from the retinal pigment epithelium layer that edge tilts, by retinal pigment epithelium
Layer (black gray expandable) is gently removed with choroid layer.
(5) the RPE cell of removing is gently transferred to and 2-4ml RPE has been added to cultivate by RPE culture medium rinse 1ml pipette tips 2-3 times
In the 15ml centrifuge tube of base, with 2000rpm, 5min centrifugation.Supernatant is abandoned after centrifugation to the greatest extent, it is light that 4-6ml RPE culture medium is added
Weight hangs cell；1, T25 culture bottle is taken, the cell of resuspension is moved in T25 culture bottle.
(6) it is put into incubator and is cultivated, changed the liquid once every 2 days；
(7) cell is taken out after culture to 240h, takes pictures, digests and count.
Using the quantity of the source of people retinal pigment epithelium of present invention digestion dissociating method removing, as shown in Figure 3.It adopts
The source of people retinal pigment epithelium culture amount removed with present invention digestion dissociating method, as shown in Figure 4.
The quantity and motility rate of the RPE cell obtained through above step, are shown in following table 2.
Table 2: the RPE cell obtained by 1 method culture of the embodiment of the present invention
Embodiment 2: the prior art, that is, traditional source of people retinal pigment epithelium (RPE) digests dissociating method
Embodiment 2 and embodiment 1 synchronize operation.
According to the identical source of people retinal pigment epithelium culture medium of recipe configuration in the table 1 in embodiment 1.
With reference to the prior art, source of people retinal pigment epithelium is dissociated in digestion according to the following steps:
(1) sealed membrane for the 15ml centrifuge tube for filling ocular tissue is opened in Biohazard Safety Equipment, it will with 1ml pipettor
Ocular tissue is sucked out, and is put into two holes of the DPBS (CTS) for 12 orifice plates being ready to complete and cleans, cleans 3-5s every time.
(2) ocular tissue is put into the 100mm culture dish for having added about 10ml DPBS (CTS), under Stereo microscope, trimming
Excess surface tissue is removed, the ocular tissue after trimming is moved into the 100mm culture dish that another fills about 10ml DPBS (CTS),
Along cornea and prosthomere is cut off at edge of cornea, eyecup is then gently squeezed, removes white nerve cell film layer.
(3) eyecup is cut off along marginal position to centre, is put into the separation enzyme solutions of 12 orifice plates after being cut into petal-shaped,
It is placed in 37.0 DEG C of carbon dioxide incubator and digests, digestion time is 50 ± 15min.
(4) after digesting, eyecup is put into the 100mm culture dish that the 3rd fills about 10ml DPBS (CTS), body formula
Under microscope, with two tissue clamps from the retinal pigment epithelium layer that edge tilts, by retinal pigment epithelium
Layer (black gray expandable) is gently removed with choroid layer.
(5) the RPE cell of removing is gently transferred to and 2-4ml RPE has been added to cultivate by RPE culture medium rinse 1ml pipette tips 2-3 times
In the 15ml centrifuge tube of base, with 2000rpm, 5min centrifugation.Supernatant is abandoned after centrifugation to the greatest extent, it is light that 4-6ml RPE culture medium is added
Weight hangs cell；1, T25 culture bottle is taken, the cell of resuspension is moved in T25 culture bottle.
(6) it is put into incubator and is cultivated, changed the liquid once every 2 days；
(7) cell is taken out after culture to 240h, takes pictures, digests and count.
Using the quantity of the source of people retinal pigment epithelium of art methods removing, as shown in Figure 5.Using existing
The source of people retinal pigment epithelium culture amount of technical method removing, as shown in Figure 6.
By 2 above step of embodiment, summarize data obtained as above, carries out data analysis.
Table 3: using the RPE cell of 2 method culture of embodiment
Eyecup is cut into petal-shaped form before enzymic digestion, is put into enzyme and digests by traditional method.And the present invention is mentioned
The method of confession trims eyecup before enzymic digestion, keeps the integrality of eyecup, then forms the inside and outside anti-cap overturn
Minor structure, RPE cellular layer is external, and non-RPE cellular layer is internal, is put into enzyme and digests.Compared with prior art, the present invention is in enzyme
Middle required time is reduced, and is reduced the loss function of RPE cellular layer, is reduced the digestible degree of non-RPE cell, maintain RPE cell
The integrality of layer provides convenience to the removing of later period RPE cellular layer, improves the acquisition of RPE cell, while decreasing non-
The pollution of RPE cell.
Can be obtained by above data: by identical training method, the present invention cultivates the RPE primary cell amount obtained and mentions
Height, and functionally and indistinction.
Comparison as it can be seen that embodiment 1 be using the method for the present invention obtain source of people retinal pigment epithelium quantity and
Culture amount is all remarkably higher than the prior art i.e. 2 method of embodiment.
Protection content of the invention is not limited to above embodiments.Under the spirit and scope without departing substantially from present inventive concept,
Various changes and advantages that will be apparent to those skilled in the art are all included in the present invention, and are with appended claims
Protection scope.
Claims (9)
1. a kind of digestion dissociating method of retinal pigment epithelium primary tissue, which is characterized in that it is comprising steps of trimming
Eyecup keeps the integrality of eyecup, eyecup is overturn, and makes the overturning of retinal pigment epithelium layer towards outside, then places into
It is digested in separation enzyme solutions, collecting after cell is cultivated can be obtained the retinal pigment epithelium.
2. the method as described in claim 1, which is characterized in that described method includes following steps:
(1) in vitro eyeball tissue outer surface excess tissue is removed, cuts off ocular tissue from cornea 2mm；
(2) eyecup is trimmed, ensures eyecup integrality；
(3) eyecup is gently squeezed, will be overturn inside and outside retinal pigment epithelium layer, non-RPE cellular layer is inside and outside inside, formation
The anti-cap sequence of overturning；
(4) eyecup after overturning being put into separation enzyme solutions, retinal pigment epithelium layer comes into full contact with separation enzyme solutions,
It is digested；
(5) after digesting, then with angle tweezer retinal pigment epithelium layer is removed manually；
(6) retinal pigment epithelium for removing abovementioned steps, which is transferred in culture bottle, cultivates, and obtains described
Retinal pigment epithelium.
3. the method as described in claim 1, which is characterized in that the retinal pigment epithelium is eyeball tissue source
Source of people retinal pigment epithelium.
4. the method as described in claim 1, which is characterized in that in step (2), the trimming eyecup are as follows: cut along cornea 2mm
Widen the view tissue, is cut off along ocular tissue's outer diameter, removal vitreum, neural confluent monolayer cells, cornea, iris and lens tissue.
5. the method as described in claim 1, which is characterized in that in step (4), the temperature of the digestion is 35 DEG C~39 DEG C；
The time of the digestion is 10min~65min.
6. the method as described in claim 1, which is characterized in that in step (6), the culture bottle is cell culture T25 culture
Bottle；The inoculum concentration of the retinal pigment epithelium is an eyeball tissue/bottle.
7. the method as described in claim 1, which is characterized in that in step (6), the time cultivated the cells is 230-
250 hours.
8. such as described in any item methods of claim 1 to 7, which is characterized in that the method also includes step (7): will walk
Suddenly the retinal pigment epithelium obtained after (6) culture is digested, is counted, is frozen.
9. digesting the retinal pigment epithelium that dissociating method is prepared as described in a kind of any one such as claim 1 to 8
Primary tissue.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602631A (en) * | 2013-11-11 | 2014-02-26 | 苏州瑞奇生物医药科技有限公司 | Separation culture method of human fetal retinal pigment epithelial cells |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602631A (en) * | 2013-11-11 | 2014-02-26 | 苏州瑞奇生物医药科技有限公司 | Separation culture method of human fetal retinal pigment epithelial cells |
Non-Patent Citations (1)
| Title |
|---|
| 龚凌等: "姜黄素对培养的人胚胎视网膜色素上皮细胞增殖活性的影响", 《眼科学报》 * |
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