CN108998413A - A kind of infant rats retinal pigment epithelium isolated culture method - Google Patents
A kind of infant rats retinal pigment epithelium isolated culture method Download PDFInfo
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- CN108998413A CN108998413A CN201810622406.1A CN201810622406A CN108998413A CN 108998413 A CN108998413 A CN 108998413A CN 201810622406 A CN201810622406 A CN 201810622406A CN 108998413 A CN108998413 A CN 108998413A
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Abstract
The invention discloses a kind of infant rats retinal pigment epithelium isolated culture methods；The following steps are included: (1) takes rat eye 0.02mg/mL Dispase collagenase treatment；(2) eyeball is dissected, removal cornea, crystal and tela chorioidea separate retina, and it is cut into retinograph, retinograph is put into incubator and is incubated for, after incubation, it flows retinograph in culture dish, retinal epithelial cells piece is promoted to separate with retinograph；(3) the retinal epithelial cells piece after separation is drawn, is cleaned in the culture dish containing PBS；(4) the retinal epithelial cells piece after cleaning is transferred in the new culture dish containing PBS, pancreatin digestion, piping and druming is added, then plant in Tissue Culture Dish, is placed in 37 DEG C, 5%CO2It is cultivated in incubator.Compared with prior art, the method for the present invention is polluted with no heteroproteose cell, and without artificial mechanically decoupled operation, process is simple, the short feature of disengaging time.
Description
Technical field
The present invention relates to cells to be separately cultured technical field, especially a kind of infant rats retinal pigment epithelium point
From cultural method.
Background technique
Retinal pigment epithelium (RPE cell) is the cell monolayer outside retina, because of its polygon form and born of the same parents
Melanin abundant in matter and gain the name, they play important function in the maintenance and self-renewing of retina cell, such as: gulping down
Bite the photoreceptor cell outer segment disk film that digestion falls off；Promote the regeneration of 11-cis retinal in view circulation；Secretory nerve nutrition because
Son；Participation forms retina-vascular barrier etc..These physiological functions of RPE cell are considered and related ophthalmology disease extremely
Occur it is closely related, such as age-related macular degeneration (age-related macular degeneration, AMD) He Laibai
Family name's congenital amaurosis disease (Leber congenital amaurosis, LCA) etc..
RPE cell has polarity, and basilar memebrane is connected with Bruch film constitutes Bruch film inner layer, top microvillus and sense
Photo-cell acromere is connected.RPE cell basal surface and Bruch film are completely embedded in adult rat, RPE cell microvillus and photosensitive
Cell acromere is completely embedded, these become difficult the separation of RPE cell.Since neonate rat (after birth in 2 weeks) is felt
Photo-cell acromere is not developed also completely, is easily isolated with RPE cell.Therefore most methods were selected in this period
It is interior, but also have the report of the RPE cell isolation method of adult rat.At present have been reported using papain (papain),
Clostridiopetidase A (collagenase) separates the RPE cell of neonate rat with a variety of methods such as Dispase dispase.These are existing
It is all made of mechanical means from the method for separating RPE cell in neonate rat to remove RPE cell from retina, operation is taken
Between it is long, difficulty is big, and be easy to cause retina relevant cell pollute.
In addition, what these methods used is pigment rat, since its RPE cell contains melanin, convenient for it is mechanically decoupled when
Identify RPE cell and retina.The limitation of these methods is more obvious for albefaction rat, due to the RPE of albefaction rat
Cell is free of pigment, it is difficult to separate with retinal field, there is presently no the reports for separating newborn white mouse RPE cell.
Summary of the invention
The present invention is insufficient for existing methods, and then provides a kind of infant rats retinal pigment epithelium separation training
The method of supporting.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of infant rats retinal pigment epithelium isolated culture method, comprising the following steps:
(1) rat eye 0.02mg/mL Dispase collagenase treatment is taken；
(2) eyeball is dissected, removal cornea, crystal and tela chorioidea separate retina, and be cut into retinograph, will regard
Caul sheet is put into incubator and is incubated for, and after incubation, gently shaking culture dish flows retinograph in culture dish, promotes view
Nethike embrane epithelial cell piece is separated with retinograph；
(3) the retinal epithelial cells piece after separation, cleaning are drawn；
(4) the retinal epithelial cells piece after cleaning is transferred in the new culture dish containing PBS, and pancreatin digestion is added, blows
It beats, then plants in Tissue Culture Dish, be placed in 37 DEG C, 5%CO2It is cultivated in incubator.
In an embodiment of the invention, step (1) rat is albefaction rat, retinal pigment epithelium
Non-pigment.It should be noted that method of the invention is applied equally to pigment rat, only for being difficult to at conventional method
The albefaction rat of reason is more applicable.
In an embodiment of the invention, step (1) rat is newborn 10 days rats.
In an embodiment of the invention, rat is taken off neck to put to death, takes out eyeball under aseptic condition.
In an embodiment of the invention, in step (1), rat eye first with penicillin containing 100U/mL and
The PBS of 100mg/mL streptomysin is rinsed, then with 0.02mg/ml Dispase enzymic digestion.
Penicillin and streptomysin are added in cell culture fluid and PBS buffer solution, is commonly called as " dual anti-".Penicillin is mainly to leather
Blue positive bacteria is effective, and streptomysin is mainly effective to gram-negative bacteria.Both antibiotic, which are added, can prevent most bacteriums dirts
Dye.
Dispase enzyme Chinese is neutral proteinase, also referred to as dispase, is a kind of nonspecific metalloprotein
Enzyme is used to the vitellophag epimatrix from various different tissues or organ, discharges and prepares primary unicellular.
Enzyme of proteolysis, as trypsase, clostridiopetidase A and pronase all come dispersion tissue, classification carefully by term
Born of the same parents, but they often injure cell, and unstable in culture, in some instances it may even be possible to introduce mycoplasma contamination.Dispase is just without this
A problem will not bring injury to cell membrane, and because it comes from bacterium, therefore will not introduce mycoplasma or any zoosis
Poison；The difference of temperature, pH, serum composition etc. influences very little to Dispase.Dispase its activity after diluting just substantially reduces,
This also facilitates subsequent culture.
In an embodiment of the invention, in step (1), with the item of 0.02mg/mL Dispase collagenase treatment
Part is: 37 DEG C digest 20~30 minutes.
In an embodiment of the invention, in step (2), dissection eyeball is in (the as volume containing 10v/v%FBS
Percentage composition) DMEM/F12 culture medium in carry out.
Retinograph is put into incubator in step (2) and is incubated for, possible principle be because retinal tissue it is very soft,
Easily crimped after separation or after being cut into sheet, but in contrast hard is a little for RPE cellular layer, after incubation retina volume together and
RPE can be separated since relative harder is not easy to crimp.
In an embodiment of the invention, in step (2), the retina after separation be cut into retinograph be use with
What lower method carried out: being directly cut into small pieces with micro- eye scissors, micro- eye scissors use elbow structure.
In an embodiment of the invention, in step (2), retinograph, which is put into the condition being incubated in incubator, is:
It is incubated for 20~30 minutes in 37 DEG C of incubators.
In an embodiment of the invention, in step (2), the method for flowing retinograph in culture dish is:
Retinograph is flowed in culture dish by culture dish is gently shaken clockwise or counterclockwise.
In an embodiment of the invention, in step (2), after incubation, flow retinograph in culture dish
After dynamic, whether separated completely with body formula sem observation, is further continued for being incubated for 10 minutes if not being kept completely separate.
In an embodiment of the invention, isolated retinal pigment epithelium is collected and shifted in step (3)
The method of piece is to be drawn and shifted with the 10uL pipettor with 10uL suction nozzle.
In an embodiment of the invention, in step (3), cleaning is cleaned in the culture dish containing PBS.
In an embodiment of the invention, in step (4), the condition that pancreatin digestion is added is: with 0.25% pancreatin
37 DEG C digest 1~2 minute.
In an embodiment of the invention, in step (4), the cell seeding density planted in Tissue Culture Dish is
15000~18000cells/cm2。
In an embodiment of the invention, after the completion of step (4), every 2 days one subcultures of replacement.
In an embodiment of the invention, 10 days SD rat retina pigment epithelial cells point of the new life of simple and fast
From cultural method, comprising the following steps:
(1) newborn 10 days SD rats take off neck and put to death, and take out eyeball under aseptic condition, with penicillin containing 100U/mL and
The PBS of 100mg/mL streptomysin is rinsed, and is put into 37 DEG C of 0.02mg/mL Dispase enzyme and is digested 20~30 minutes.
(2) eyeball is dissected in the DMEM/F12 culture medium containing 10v/v%FBS, removes cornea, the groups such as crystal and choroid
It knits, separate retina and is cut into small pieces, be put into 37 DEG C of incubators and be incubated for 20~30 minutes.By clockwise or inverse after incubation
Clockwise, which gently shakes culture dish, flows retinograph in culture dish, promotes retinal epithelial cells and view UF membrane.
Whether body formula microscopic observation separates completely, is further continued for being incubated for 10 minutes if not being kept completely separate.
(3) the retinal epithelial cells piece after separation is drawn with pipettor, is transferred in the 3cm culture dish containing PBS and cleans,
The retinal epithelial cells piece after cleaning is drawn again and is transferred in new 3cm culture dish.
(4) 0.25% pancreatin is added, 37 DEG C digest 1~2 minute, terminate and digest and blow and beat, by 16000cells/cm2Kind
It plants in Tissue Culture Dish, is placed in 37 DEG C, 5%CO2It is cultivated in incubator.Every 2 days later one subcultures of replacement.
Compared with prior art, the advantages and beneficial effects of the present invention are:
Infant rats retinal pigment epithelium isolated culture method provided by the present invention is a kind of simple and fast
The method for separating infant rats retinal pigment epithelium.By separating retina after enzymic digestion and being cut into small pieces, through training liquid
After being incubated for separation, it can cultivate and obtain a large amount of retinal pigment epitheliums, no heteroproteose cell pollution, without artificial mechanically decoupled behaviour
Make, process is simple, shortens disengaging time.
Detailed description of the invention
Fig. 1 be SD rat retina pigment epithelial cell separation specific steps schematic diagram (Figure 1A be digest enzymic digestion after
Isolated retina；Figure 1B is the retina for being cut into sheet；Fig. 1 C is the retinal epithelial cells with view UF membrane, wherein arrow
First 1 is designated as retinal pigment epithelium, and arrow 2 is designated as retina).
Fig. 2 is the SD rat retina pigment epithelial cell of in vitro culture 7d.
Fig. 3 be DA rat retina pigment epithelial cell separation specific steps schematic diagram (Fig. 3 A be digest enzymic digestion after
Isolated retina；Fig. 3 B is the retina for being cut into sheet；Fig. 3 C is the retinal epithelial cells with view UF membrane, wherein arrow
First 1 is designated as retinal pigment epithelium, and arrow 2 is designated as retina).
Fig. 4 is the DA rat retina pigment epithelial cell of in vitro culture 7d.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
The separation and culture of newborn 10 days SD rat retina pigment epithelial cells.
(1) newborn 10 days SD rats take off neck and put to death, and take out eyeball under aseptic condition, with penicillin containing 100U/mL and
The PBS of 100mg/mL streptomysin is rinsed, and is put into 37 DEG C of 0.02mg/mL Dispase enzyme and is digested 30 minutes.
(2) eyeball is dissected in the DMEM/F12 culture medium containing 10v/v%FBS, removes cornea, the groups such as crystal and choroid
It knits, separate retina and is cut into small pieces, be put into 37 DEG C of incubators and be incubated for 30 minutes (Figure 1A and B).Up time is pressed after incubation
Culture dish is gently shaken in direction to needle (or counterclockwise) flows retinograph in culture dish, promotes retinal epithelial cells and view
UF membrane (Fig. 1 C).Whether body formula microscopic observation separates completely, is further continued for being incubated for 10 minutes if not being kept completely separate.
(3) the retinal epithelial cells piece after separation is drawn with pipettor, is transferred in the 3cm culture dish containing PBS and cleans,
The retinal epithelial cells piece after cleaning is drawn again and is transferred in new 3cm culture dish.
(4) 0.25% pancreatin is added, 37 DEG C digest 2 minutes, terminate and digest and blow and beat, by 16000cells/cm2Plant in
In Tissue Culture Dish, 37 DEG C are placed in, 5%CO2It is cultivated in incubator.Every 2 days later one subcultures of replacement.
The SD rat retina pigment epithelial cell of in vitro culture 7d is as shown in Figure 2.
Embodiment 2
The separation and culture of newborn 10 days DA rat retina pigment epithelial cells.
(1) newborn 10 days DA rats take off neck and put to death, and take out eyeball under aseptic condition, with penicillin containing 100U/mL and
The PBS of 100mg/mL streptomysin is rinsed, and is put into 37 DEG C of 0.02mg/mL Dispase enzyme and is digested 30 minutes.
(2) eyeball is dissected in the DMEM/F12 culture medium containing 10v/v%FBS, removes cornea, the groups such as crystal and choroid
It knits, separate retina and is cut into small pieces, be put into 37 DEG C of incubators and be incubated for 30 minutes (Fig. 3 A and B).Up time is pressed after incubation
Needle or counter clockwise direction gently shake culture dish and flow retinograph in culture dish, promote retinal epithelial cells and retina
It separates (Fig. 3 C).Whether body formula microscopic observation separates completely, is further continued for being incubated for 10 minutes if not being kept completely separate.
(3) the retinal epithelial cells piece after separation is drawn with pipettor, is transferred in the 3cm culture dish containing PBS and cleans,
The retinal epithelial cells piece after cleaning is drawn again and is transferred in new 3cm culture dish.
(4) 0.25% pancreatin is added, 37 DEG C digest 2 minutes, terminate and digest and blow and beat, by 16000cells/cm2Plant in
In Tissue Culture Dish, 37 DEG C are placed in, 5%CO2It is cultivated in incubator.Every 2 days later one subcultures of replacement
The DA rat retina pigment epithelial cell of in vitro culture 7d is as shown in Figure 4.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (10)
1. a kind of infant rats retinal pigment epithelium isolated culture method, which comprises the following steps:
(1) rat eye 0.02mg/ml Dispase collagenase treatment is taken；
(2) eyeball is dissected, removal cornea, crystal and tela chorioidea separate retina, and be cut into retinograph, by retina
Piece is put into incubator and is incubated for, and after incubation, flows retinograph in culture dish, promote retinal epithelial cells piece with
Retinograph separation；
(3) the retinal epithelial cells piece after separation is drawn, is cleaned in the culture dish containing PBS；
(4) the retinal epithelial cells piece after cleaning is transferred in the new culture dish containing PBS, and pancreatin digestion, piping and druming is added, then
It plants in Tissue Culture Dish, is placed in incubator and cultivates.
2. a kind of infant rats retinal pigment epithelium isolated culture method according to claim 1, feature exist
In step (1) rat is albefaction rat.
3. a kind of infant rats retinal pigment epithelium isolated culture method according to claim 1, feature exist
In step (1) rat is newborn 10 days rats.
4. a kind of infant rats retinal pigment epithelium isolated culture method according to claim 1, feature exist
In in step (1), rat eye is first rinsed with the PBS of penicillin containing 100U/mL and 100mg/mL streptomysin, then uses 0.02mg/
Ml Dispase enzymic digestion.
5. a kind of infant rats retinal pigment epithelium isolated culture method according to claim 1, feature exist
In in step (2), dissection eyeball is carried out in the DMEM/F12 culture medium containing 10v/v%FBS.
6. a kind of infant rats retinal pigment epithelium isolated culture method according to claim 1, feature exist
In, in step (2), be cut into retinograph using following methods carry out: be directly cut into small pieces with micro- eye scissors.
7. a kind of infant rats retinal pigment epithelium isolated culture method according to claim 1, feature exist
In in step (2), retinograph, which is put into the condition being incubated in incubator, is: being incubated for 20~30 minutes in 37 DEG C of incubators.
8. a kind of infant rats retinal pigment epithelium isolated culture method according to claim 1, feature exist
In in step (2), the method for flowing retinograph in culture dish is: by gently shaking culture clockwise or counterclockwise
Ware flows retinograph in culture dish.
9. a kind of infant rats retinal pigment epithelium isolated culture method according to claim 1, feature exist
In in step (4), the condition that pancreatin digestion is added is: being digested 1~2 minute with 37 DEG C of 0.25% pancreatin.
10. a kind of infant rats retinal pigment epithelium isolated culture method according to claim 1, feature exist
In, after the completion of step (4), every 2 days one subcultures of replacement.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011063005A2 (en) * | 2009-11-17 | 2011-05-26 | Advanced Cell Technology, Inc. | Methods of producing human rpe cells and pharmaceutical preparations of human rpe cells |
| 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 |
| CN106434531A (en) * | 2016-11-30 | 2017-02-22 | 江苏艾尔康生物医药科技有限公司 | Human retinal pigment epithelial cell separation and cryopreservation method |
-
2018
- 2018-06-15 CN CN201810622406.1A patent/CN108998413A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011063005A2 (en) * | 2009-11-17 | 2011-05-26 | Advanced Cell Technology, Inc. | Methods of producing human rpe cells and pharmaceutical preparations of human rpe cells |
| 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 |
| CN106434531A (en) * | 2016-11-30 | 2017-02-22 | 江苏艾尔康生物医药科技有限公司 | Human retinal pigment epithelial cell separation and cryopreservation method |
Non-Patent Citations (2)
| Title |
|---|
| C W CHANG ET.AL: "An Improved Method for Isolation and Culture of Pigment Epithelial Cells From Rat Retina", 《CURR EYE RES》 * |
| 潘求真，岳才军主编: "《 细胞工程[M].》", 31 July 2009, 哈尔滨：哈尔滨工程大学出版社 * |
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