CN106801039A - A kind of construction method in Human RPE Cells in Vitro storehouse - Google Patents
A kind of construction method in Human RPE Cells in Vitro storehouse Download PDFInfo
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- CN106801039A CN106801039A CN201611153058.5A CN201611153058A CN106801039A CN 106801039 A CN106801039 A CN 106801039A CN 201611153058 A CN201611153058 A CN 201611153058A CN 106801039 A CN106801039 A CN 106801039A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5058—Neurological cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The present invention relates to a kind of construction method in Human RPE Cells in Vitro storehouse, step includes respectively：Collection contributes people eyeball, separates and obtains Human RPE Cells in Vitro, Secondary Culture, the quality testing of Human RPE Cells in Vitro, freeze He Jianku, and the recovery of the Human RPE Cells in Vitro that will be frozen when using can be used for clinical injection.Step operation of the present invention is simple, the Human RPE Cells in Vitro purity that obtain, cultivate, freezes is high, multiplication capacity is strong, recovery efficiency high, the foundation of cell bank is according to HLA distribution type, by may be directly applied to clinical injection after the cell recovery of quality testing and HLA distribution type, production, preservation, the transport of cell preparation are solved the problems, such as, when patient needs to be transplanted using Human RPE Cells in Vitro, the Human RPE Cells in Vitro of clinical applicable HLA distribution type can be directly provided.
Description
Technical field
The present invention relates to a kind of construction method in Human RPE Cells in Vitro storehouse, belong to biological technical field.
Background technology
Retinal degeneration is the Etiological for causing irreversible diseases causing blindness, wherein mainly including age related
Macular degeneration, retinal pigment degeneration, Stargardt diseases.Wherein age-related macular degeneration patients, the whole world about 3,000
Ten thousand to 5 thousand ten thousand, and patients with retinitis pigmentosa about 1,000,000.The age-related macular degeneration patients of current China about surpass
20,000,000 people are crossed, and is expected to be doubled in the year two thousand fifty.
Because Retinal degeneration is mainly shown as macular area retinal pigment epithelium (Retinal Pigment
Epithelium, RPE) damage, therefore regenerative medicine RPE cells substitute transplantation treatment turn into Therapy study main side
To.The RPE cell transplantations in various sources are had reported so far, mainly have autologous patient RPE cell transplantations, pigmented epithelium of iris,epithelium pigmentosum iridis thin
Born of the same parents (Iris Pigment Epithelium, IPE) transplanting, embryo and adult's RPE cell transplantations, and embryonic stem cell
(Embryonic Stem Cells, ESCs) and induced multi-potent stem cell (induced pluripotent stem cells,
IPSCs) the RPE cell transplantations in source.These cell transplantations show clear and definite therapeutic action in zoopery, but again
There is respective defect.Autologous patient RPE transfer operations are complicated, there is PVR complication, the problem of AMD recurrences；IPE is to certain
A little retinal degeneration models have therapeutic action, but the express spectra research to IPE and RPE shows that IPE lacks rhodopsin and follows
Some of ring key enzyme, can not completely substitute the physiological function of RPE, and correlative study in recent years is also reduced increasingly；Embryo
There is dispute of ethic in the clinical practice of stem cell, because of potential oncogenicity, its security also makes us denouncing all the time；IPSCs was building
Potential proto-oncogene is imported into journey, retrovirus is integrated into host genome can also increase the risk of tumorigenesis, most
Near research also demonstrate that iPSCs has teratoma formability higher than embryonic stem cell.Although studies have reported that reduce or
Remove the exogenous transcriptional factor completely, or by micromolecular compound, or solved by the method removed again after importing
Problem.But no matter a kind of that method need further checking to iPSCs into preclinical security.
Research shows that people's RPE cells have immunogenicity small, and the significant specific immune response of T cell is not caused, may be used also
To suppress t cell activation, it is to avoid the use of immunodepressant；With great in-vitro multiplication ability, people's eyeball can be with
For treating hundreds AMD patient, materials limitation, financial burden and the ethics arguement to people's RPE cell clinical practices are alleviated；
People RPE cells can secrete trophic factors in vivo, and Synaptic junction is formed with host retina；In tumorigenicity risk substantially
Better than the RPE cells of source of human stem cell；The advantages of various RPE distinctive functional markers can be expressed, is retinal pigment
Epithelial cell transplantation substitute treats one of most promising cell derived.
The content of the invention
The invention aims to solve the above problems, there is provided a kind of people's view for according to HLA distribution type build storehouse
The construction method in membranochromic pigments epithelial cell storehouse.
The present invention is adopted the following technical scheme that：A kind of construction method in Human RPE Cells in Vitro storehouse, including it is as follows
Step：
（1）People's eyeball is contributed in collection：ABO/Rh Blood groupings, the detection of HLA partings, microorganism immune detection, eye are carried out to donor
Ball departs from live body to be completed in 12 hours to collection；
（2）Separate and obtain Human RPE Cells in Vitro：The epibulbar musculature that will be gathered is clear with DPBS after removing
Wash eyeball 2-3 times, each 5-8 minutes, excision cornea, iris, and crystalline lens and vitreum are removed, eyecup is extruded, by view
Film is removed, and separates layer of retina,pigment epithelium and choroid layer, and the layer of retina,pigment epithelium of separation is placed in into trypsase 1-
2ml digests 10-15min at 37 DEG C, adds 10ml nutrient solutions to terminate digestion, and centrifugation abandons supernatant, with the resuspended acquisition of nutrient solution
Retinal pigment epithelium is inoculated with；
（3）Secondary Culture：The cell for obtaining is pressed into 3-6 × 104/ cm2 adds 5% blood platelet in being inoculated in six orifice plates or blake bottle
Lysate without animal originality, the culture medium without serum composition carries out Secondary Culture, and obtains the people's view after Secondary Culture
Membranochromic pigments epithelial cell；
（4）Detection Human RPE Cells in Vitro：The culture medium supernatant of culture 3 days is drawn, for the secreting function inspection of cell
Survey, by the Human RPE Cells in Vitro of Secondary Culture carry out respectively after digestion process Sterility testing, detection of mycoplasma,
Viral diagnosis, streaming purity detecting, VEGF and PEDF secreting functions, the detection of MHC molecule expression quantity, HLA partings, cell count and
Vitality test, ability of cell proliferation detection, Apoptosis cycle detection and the detection of Tumor formation, whether detection cell meets is built storehouse
Quality standard；
（5）Freeze Human RPE Cells in Vitro：By through the qualified Human RPE Cells in Vitro of quality testing, to passage
The cell of culture is digested, and according to cell counts, the cell suspension centrifugation for drawing 5 × 105 cells is abandoned supernatant, added
Re-suspended cell after 0.45ml platelet lysates liquids and 0.05ml dimethyl sulfoxide (DMSO) mixed liquors, and will together freeze in suction cryopreservation tube
Deposit pipe to be put into program temperature reduction box, move into -80 DEG C of refrigerators rapidly；After 24 hours, preserved for a long time in immigration liquid nitrogen；
（6）Build storehouse：The Human RPE Cells in Vitro of acquisition is set up the cellular informatics archives for being available for retrieval, that is, constructs people
The cell bank of retinal pigment epithelium；
（7）The recovery of the Human RPE Cells in Vitro that will be frozen when using can be used for clinical injection.
Further, the step（3）In Secondary Culture comprise the following steps：
（a）In six orifice plates or blake bottle, it is placed in 37 DEG C, cultivates in the incubator that CO2 contents are 5%, example of spatial compartmentalizationis,
Non-adherent cell is discarded, more renews culture medium once within every 3 days later；
（b）Treat that cell growth is merged or 1-2 weeks to 80%, add trypsase 1-2ml to be disappeared in six orifice plates or blake bottle
Change；
（c）Six orifice plates or blake bottle are put into 5-8min in 37 DEG C of incubators, observation of cell is rounded under inverted microscope, uses palm
Gently patting blake bottle makes cell and tissue block digest completely, in being diluted with the culture medium or DPBS buffer solutions of 10ml
Only, supernatant is abandoned in the cell suspension centrifugation that will be washed down, suction;
（d）To cell count is carried out with thrombocytometer after adding 4ml culture mediums in centrifuge tube, 3-6 is pressed according to count results
× 104/cm2 is inoculated in six orifice plates or blake bottle, supplies culture medium.
Further, the step（4）The method of middle Human RPE Cells in Vitro pre-treatment is：It is slow with 2ml DPBS
Human RPE Cells in Vitro after fliud flushing washing Secondary Culture 2-3 times, washing lotion is abandoned in suction, and trypsase 1-2ml is digested,
Termination is diluted with 10ml DPBS buffer solutions, is blown and beaten repeatedly, the cell in six orifice plates or blake bottle is cleaned up, will be clear
Wash in the cell suspension immigration centrifuge tube for getting off, be centrifuged 5-8 minutes under 1000-2000 revs/min of speed conditions, centrifugation knot
Shu Hou, suction abandon supernatant, in centrifuge tube add 4-5ml contain 5% platelet lysates liquid without animal originality, without serum composition
Culture medium after carry out cell count with thrombocytometer.
Further, the step（7）After Human RPE Cells in Vitro is taken out from liquid nitrogen during middle recovery at once
It is placed in 37 DEG C of water-baths and quickly dissolves, after after frozen stock solution thawing, celliferous 1ml frozen stock solutions is put into 10ml without heparin
Vitreous chamber perfusion liquid, 1000-2000 revs/min is centrifuged 5-8 minutes, supernatant discarded after centrifugation.
Further, the step（7）After Human RPE Cells in Vitro is taken out from liquid nitrogen during middle recovery at once
It is placed in 37 DEG C of water-baths and quickly dissolves, after after frozen stock solution thawing, celliferous 1ml frozen stock solutions is put into 10ml 4U/ containing heparin
In the vitreous chamber perfusion liquid of ml, 1000-2000 revs/min is centrifuged 5-8 minutes, supernatant discarded after centrifugation.
The present invention has advantages below：Step operation of the present invention is simple, on the human retinal pigment for obtain, cultivate, freezing
Chrotoplast purity is high, multiplication capacity strong, recovery efficiency high, the foundation foundation HLA distribution type of cell bank, by quality testing and HLA
Clinical injection is may be directly applied to after the cell recovery of distribution type, production, preservation, the transport of cell preparation is solved the problems, such as,
When patient needs to be transplanted using Human RPE Cells in Vitro, people's view of clinical applicable HLA distribution type can be directly provided
Membranochromic pigments epithelial cell.
Brief description of the drawings
Fig. 1 is the donation people's eyeball obtained in the present invention.
Fig. 2 is separation layer of retina,pigment epithelium and choroid layer in the present invention, wherein：1-cornea；2-retina；
3-choroid；4-retinal pigment epithelium.
Fig. 3 is the retinal pigment epithelium that acquisition is centrifuged in the present invention after Trypsin Induced.
Fig. 4 is retinal pigment epithelium P1 of the invention for photo.
Fig. 5 is retinal pigment epithelium purity flow testing result of the invention.
Fig. 6 is active flow cytometer detection knot after retinal pigment epithelium of the invention is recovered with four kinds of different resuscitation fluids
Really.
Specific embodiment
Below in conjunction with accompanying drawing, the invention will be further described.
A kind of construction method in Human RPE Cells in Vitro storehouse, method is as follows：
Donor is carried out ABO/Rh Blood groupings, HLA partings detection, microorganism immune detection, physical examination HIV, HBV, HCV, syphilis,
Medical history and family history to donor and its relatives carry out investigation confirmation not in contact with teratogen excessively, without heredity medication history.Confirm donor label
The basic conditions such as administration's Informed Consent Form.
It is the aborted fetus that gestational age is 95 days that this example contributes people's eyeball, induced abortion, free from infection, is become without retinal pigment
Property and ocular tumor family history.Aborted fetus is rinsed with the other DPBS of clinical grade, the blood and dirt on surface is washed away.Use high pressure
The ophthalmologic operation of sterilizing is cut, and takes out eyeball along wall of eyeball, and the muscle around wall of eyeball is cut off in the other DPBS of clinical grade
Tissue（As shown in Figure 1）.Eyeball is cleaned with the other DPBS of clinical grade 2 times, 5 minutes every time.At corneal limbus 1-2mm, angle is cut off
Film, iris, and crystalline lens and vitreum are removed.Extruding eyecup, gently discharges retina.Cut with ophthalmologic operation and cut eyecup
Into petal-shaped, pave（1 is cornea shown in accompanying drawing 2；2 is retina；3 is choroid；4 is retinal pigment epithelium）.From black
The edge of retinal pigment epithelium start, with eye surgery forceps by retinal pigment epithelium little by little with choroid layer point
From.The retinal pigment epithelium liquid-transfering gun torn is drawn in 15ml centrifuge tubes, and adds the other pancreatin of clinical grade, 37 DEG C
Digested 10 minutes in incubator.Retinal pigment epithelium tissue is blown and beaten repeatedly, and 4ml culture mediums are added after its digestion is separated, with
1000 revs/min are centrifuged 5 minutes（As shown in Figure 3）.Abandoning supernatant after centrifugation, and add 4ml nutrient solutions resuspended, it is seeded in
With in 6 standby orifice plates of clinical rank matrigel bag.
Liquid is changed within second day after inoculation, and every two days change liquid once（As shown in Figure 4）.Passed on after cell is covered with：
The digestion of 1ml clinical grades other pancreatin is added in six orifice plates per hole, is put into 37 DEG C of incubators and is digested 3 minutes.It was observed that cell becomes
After justifying and suspending, it is added immediately 10ml culture mediums and stops digestion.Culture plate is blown and beaten repeatedly, helps cell to suspend, and cell is inhaled
Enter in 15ml centrifuge tubes, 1000 revs/min are centrifuged 5 minutes.Supernatant discarded after centrifugation, adds culture medium re-suspended cell, and count.
With 3 × 105The concentration of cells/well, cell is seeded on 6 well culture plates that matrigel bag is got ready.
Hereafter cultural method and propagating method is the same.
After inoculation after 2 weeks, a large amount of typical hexagon retinal pigment epitheliums of appearance of Secondary Culture（Such as Fig. 4 institutes
Show）.After cultivating 5 weeks, the culture medium supernatant of culture 3 days is drawn, for the secreting function detection of cell.Digest 3 hole retinal colors
Plain epithelial cell about draws 6 × 106The cell suspension of individual cell is respectively at carrying out quality testing in 10 EP pipes.Quality testing exists
Medicine non-clinical study quality control procedure standard test is carried out in room, and content includes：Sterility testing, detection of mycoplasma, virus
Detection, the detection of streaming purity detecting, VEGF and PEDF secreting functions, MHC molecule expression quantity, HLA partings, cell count and vigor
Measure, ability of cell proliferation detection, Apoptosis cycle detection, Tumor formation detection, to determine that it reaches criterion of acceptability.Wherein flow
The lower retinal pigment epithelium surface protein of formula cell instrument detection culture marks the expression of Mertk and RPE65（As shown in Figure 5）
It is 96.9% to obtain cell purity（>95%, up-to-standard).
Freeze and build storehouse：The P2 for obtaining for 4-5 weeks will be cultivated to add per hole in six orifice plates for Human RPE Cells in Vitro
Enter the digestion of 1ml clinical grades other pancreatin, be put into 37 DEG C of incubators and digest 5-10 minutes.It was observed that after cell rounding and suspension,
It is added immediately the dilution of 10ml culture mediums and stops digestion.Blow and beat culture plate repeatedly, help cell to suspend, and by cell suction 15ml from
In heart pipe, count, 1000 revs/min are centrifuged 5 minutes.Supernatant discarded after centrifugation, with 1 × 106The concentration of individual/ml, cell is existed
The frozen stock solution for configuring in advance（90% platelet lysates liquid+10%DMSO）In it is resuspended.Then it is put at once in program temperature reduction box and puts
Enter -80 DEG C of refrigerators to freeze, taking-up is preserved for a long time in being put into liquid nitrogen container after 24 hours.The each person-portion human retinal pigment that will be obtained
Epithelial cell（5×105/ pipe）, preserved respectively by donor name, identification card number, admission number, HLA partings, storage date, set up
Per the computer data of the detailed archives of portion cell, the cell bank of Human RPE Cells in Vitro is set up.
Recovery：The P2 that will be frozen is placed on 37 DEG C at once after being taken out from liquid nitrogen for Human RPE Cells in Vitro cryopreservation tube
Quickly dissolved in water-bath.After after frozen stock solution thawing, celliferous frozen stock solution is respectively put into vitreums of the 10ml without heparin
Chamber perfusion liquid（BSS）, 10ml contain heparin（4U/ml）Vitreous chamber perfusion liquid（BSS）In, 10ml balanced salt solutions（PBS）、
10ml Human RPE Cells in Vitro culture mediums, 1000 revs/min are centrifuged 5 minutes.Supernatant discarded after centrifugation, uses fluidic cell
Instrument is to the retinal pigment epithelium Activity determination after cryopreservation resuscitation.Result such as Fig. 6 shows, multiple to freezing with flow cytometer
Retinal pigment epithelium Activity determination result after Soviet Union shows, vitreous chamber perfusion liquid using 10ml without heparin and
10ml balanced salt solutions（PBS）The cytoactive of recovery can meet or exceed 90%, and cytoactive is without system between two kinds of method for resuscitation
Meter learns difference（P=0.0956,n=3）.The cytoactive recovered using vitreous chamber perfusion liquids of the 10ml containing heparin 40U is substantially dropped
It is low, less than the cell recovered using vitreous chamber perfusion liquids of the 10ml without heparin（P=0.0014,n=3）.Regarded using 10ml people
The cytoactive of retinal pigment epithelial cell culture medium recovery is significantly reduced, less than using vitreous chambers of the 10ml without heparin
The cell of perfusion liquid recovery（P=0.001,n=3）, but using the human retina of Human RPE Cells in Vitro culture medium recovery
Pigment epithelial cell cannot be directly used to clinical injection, therefore the method for resuscitation of Human RPE Cells in Vitro uses 10ml not
Vitreous chamber perfusion liquid containing heparin（BSS）, 10ml contain heparin（4U/ml）Vitreous chamber perfusion liquid（BSS）Two ways.
In a word, it is of the invention without animal originality, the in-vitro separation of serum-free composition, cultivate, freeze under system, people's view
Membranochromic pigments epithelial cell has typical hexagonal configuration, and purity can reach more than 95%, solve existing retinal pigment
The not enough present situation of epithelial cell origin, and the cell bank of longer-term storage Human RPE Cells in Vitro is set up, easy to operate,
With low cost, definite ingredients and stablize, provide a large amount of cellular resources to clinical and research application.By quality testing it is qualified and
The Human RPE Cells in Vitro of HLA distribution type, can be used for clinical injection after recovery, solve the production of cell preparation, preservation,
The problem of transport.
Claims (5)
1. a kind of construction method in Human RPE Cells in Vitro storehouse, it is characterised in that：Comprise the following steps：
（1）People's eyeball is contributed in collection：ABO/Rh Blood groupings, the detection of HLA partings, microorganism immune detection, eye are carried out to donor
Ball departs from live body to be completed in 12 hours to collection；
（2）Separate and obtain Human RPE Cells in Vitro：The epibulbar musculature that will be gathered is clear with DPBS after removing
Wash eyeball 2-3 times, each 5-8 minutes, excision cornea, iris, and crystalline lens and vitreum are removed, eyecup is extruded, by view
Film is removed, and separates layer of retina,pigment epithelium and choroid layer, and the layer of retina,pigment epithelium of separation is placed in into trypsase 1-
2ml digests 10-15min at 37 DEG C, adds 8-10ml nutrient solutions to terminate digestion, and centrifugation abandons supernatant, with the resuspended acquisition of nutrient solution
Retinal pigment epithelium be inoculated with；
（3）Secondary Culture：The cell for obtaining is pressed into 3-6 × 104/cm2Being inoculated in six orifice plates or blake bottle adds 5% blood platelet to split
Solve liquid carries out Secondary Culture without animal originality, the culture medium without serum composition, and obtains the human retina after Secondary Culture
Pigment epithelial cell；
（4）Detection Human RPE Cells in Vitro：The culture medium supernatant of culture 3 days is drawn, for the secreting function inspection of cell
Survey, by the Human RPE Cells in Vitro of Secondary Culture carry out respectively after digestion process Sterility testing, detection of mycoplasma,
Viral diagnosis, streaming purity detecting, VEGF and PEDF secreting functions, the detection of MHC molecule expression quantity, HLA partings, cell count and
Vitality test, ability of cell proliferation detection, Apoptosis cycle detection and the detection of Tumor formation, whether detection cell meets is built storehouse
Quality standard；
（5）Freeze Human RPE Cells in Vitro：By through the qualified Human RPE Cells in Vitro of quality testing, to passage
The cell of culture is digested, and according to cell counts, draws 5 × 105Supernatant is abandoned in the cell suspension centrifugation of individual cell, is added
Re-suspended cell after 0.45ml platelet lysates liquids and 0.05ml dimethyl sulfoxide (DMSO) mixed liquors, and will together freeze in suction cryopreservation tube
Deposit pipe to be put into program temperature reduction box, move into -80 DEG C of refrigerators rapidly；After 24 hours, preserved for a long time in immigration liquid nitrogen；
（6）Build storehouse：The Human RPE Cells in Vitro of acquisition is set up the cellular informatics archives for being available for retrieval, that is, constructs people
The cell bank of retinal pigment epithelium；
（7）The recovery of the Human RPE Cells in Vitro that will be frozen when using can be used for clinical injection.
2. the construction method in Human RPE Cells in Vitro storehouse as claimed in claim 1, it is characterised in that：The step
（3）In Secondary Culture comprise the following steps：
（a）In six orifice plates or blake bottle, 37 DEG C, CO are placed in2Content be 5% incubator in cultivate, example of spatial compartmentalizationis abandons
Non-adherent cell is removed, more renews culture medium once within every 3 days later；
（b）Treat that cell growth is merged or 1-2 weeks to 80%, add trypsase 1-2ml to be disappeared in six orifice plates or blake bottle
Change；
（c）Six orifice plates or blake bottle are put into 5-8min in 37 DEG C of incubators, observation of cell is rounded under inverted microscope, uses palm
Gently patting blake bottle makes cell and tissue block digest completely, in being diluted with the culture medium or DPBS buffer solutions of 10ml
Only, supernatant is abandoned in the cell suspension centrifugation that will be washed down, suction;
（d）To cell count is carried out with thrombocytometer after adding 4ml culture mediums in centrifuge tube, 3-6 is pressed according to count results
×104/cm2It is inoculated in six orifice plates or blake bottle, supplies culture medium.
3. the construction method in Human RPE Cells in Vitro storehouse as claimed in claim 1, it is characterised in that：The step
（4）The method of middle Human RPE Cells in Vitro pre-treatment is：The people washed after Secondary Culture with 2ml DPBS buffer solutions regards
Washing lotion is abandoned in retinal pigment epithelial cell 2-3 times, suction, and trypsase 1-2ml is digested, and is carried out with 10ml DPBS buffer solutions dilute
Termination is released, is blown and beaten repeatedly, the cell in six orifice plates or blake bottle is cleaned up, the cell suspension that will be washed down moves into centrifugation
Guan Zhong, is centrifuged 5-8 minutes under 1000-2000 revs/min of speed conditions, and after centrifugation terminates, supernatant is abandoned in suction, in centrifuge tube
Add 4-5ml contain 5% platelet lysates liquid without using thrombocytometer after animal originality, the culture medium without serum composition
Carry out cell count.
4. the construction method in Human RPE Cells in Vitro storehouse as claimed in claim 1, it is characterised in that：The step
（7）It is placed at once after Human RPE Cells in Vitro is taken out from liquid nitrogen during middle recovery in 37 DEG C of water-baths and is quickly dissolved,
After after frozen stock solution thawing, celliferous 1ml frozen stock solutions are put into vitreous chamber perfusion liquids of the 10ml without heparin, 1000-2000
Rev/min centrifugation 5-8 minutes, supernatant discarded after centrifugation.
5. the construction method in Human RPE Cells in Vitro storehouse as claimed in claim 1, it is characterised in that：The step
（7）It is placed at once after Human RPE Cells in Vitro is taken out from liquid nitrogen during middle recovery in 37 DEG C of water-baths and is quickly dissolved,
After after frozen stock solution thawing, celliferous 1ml frozen stock solutions are put into vitreous chamber perfusion liquids of the 10ml containing heparin 4U/ml, 1000-
2000 revs/min are centrifuged 5-8 minutes, supernatant discarded after centrifugation.
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| CN108998496A (en) * | 2018-08-20 | 2018-12-14 | 北京市眼科研究所 | A kind of evaluation method of artificial light source visual system injury |
| CN110249811A (en) * | 2019-05-29 | 2019-09-20 | 甘肃农业大学 | Ensilage crushing rubs silk and is packaged integrated working rig |
| CN110249811B (en) * | 2019-05-29 | 2023-11-24 | 甘肃农业大学 | Silage crushing, silk kneading and packaging integrated operation machine |
| CN111066777A (en) * | 2019-12-19 | 2020-04-28 | 镇江雷音再生医学科技有限公司 | Corneal lens ultralow-temperature long-term storage method |
| CN114586769A (en) * | 2020-12-07 | 2022-06-07 | 精准生技股份有限公司 | Serum-free cell cryopreservation liquid for cell cryopreservation and cryopreservation method thereof |
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