CN106434531A - Human retinal pigment epithelial cell separation and cryopreservation method - Google Patents
Human retinal pigment epithelial cell separation and cryopreservation method Download PDFInfo
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- 238000005138 cryopreservation Methods 0.000 title claims abstract description 20
- 210000000844 retinal pigment epithelial cell Anatomy 0.000 title claims abstract description 19
- 238000000926 separation method Methods 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 18
- 210000004027 cell Anatomy 0.000 claims abstract description 35
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 claims abstract description 14
- 108010007093 dispase Proteins 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 13
- 238000005406 washing Methods 0.000 claims abstract description 7
- 239000007853 buffer solution Substances 0.000 claims abstract description 3
- 210000003583 retinal pigment epithelium Anatomy 0.000 claims description 41
- 230000029087 digestion Effects 0.000 claims description 20
- 239000001963 growth medium Substances 0.000 claims description 16
- 239000007789 gas Substances 0.000 claims description 15
- 210000002249 digestive system Anatomy 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 11
- 239000012531 culture fluid Substances 0.000 claims description 10
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 10
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 claims description 10
- 230000003833 cell viability Effects 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000006285 cell suspension Substances 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 229960000890 hydrocortisone Drugs 0.000 claims description 5
- 239000011550 stock solution Substances 0.000 claims description 5
- 229960003080 taurine Drugs 0.000 claims description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 4
- 210000001772 blood platelet Anatomy 0.000 claims description 4
- 239000006166 lysate Substances 0.000 claims description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 3
- 229930182816 L-glutamine Natural products 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 230000002255 enzymatic effect Effects 0.000 claims description 3
- 239000000654 additive Substances 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims description 2
- 229940024606 amino acid Drugs 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims 10
- 229910002092 carbon dioxide Inorganic materials 0.000 claims 5
- 239000001569 carbon dioxide Substances 0.000 claims 1
- 239000000790 retinal pigment Substances 0.000 claims 1
- 102000005734 Separase Human genes 0.000 abstract 4
- 108010031091 Separase Proteins 0.000 abstract 4
- 230000001079 digestive effect Effects 0.000 abstract 4
- 239000012530 fluid Substances 0.000 abstract 2
- 230000005779 cell damage Effects 0.000 abstract 1
- 208000037887 cell injury Diseases 0.000 abstract 1
- 238000004140 cleaning Methods 0.000 abstract 1
- 235000015097 nutrients Nutrition 0.000 abstract 1
- 108010019160 Pancreatin Proteins 0.000 description 8
- 229940055695 pancreatin Drugs 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 3
- 238000010009 beating Methods 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000790917 Dioxys <bee> Species 0.000 description 2
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 2
- 206010064930 age-related macular degeneration Diseases 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
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- 230000005593 dissociations Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 208000002780 macular degeneration Diseases 0.000 description 2
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- JMSOBUJWXGTTLB-OAHLLOKOSA-N (2S)-2-amino-3-[4-(4-hydroxyphenoxy)phenyl]-2,3,3-triiodopropanoic acid Chemical compound IC([C@](N)(C(=O)O)I)(C1=CC=C(C=C1)OC1=CC=C(C=C1)O)I JMSOBUJWXGTTLB-OAHLLOKOSA-N 0.000 description 1
- NWNOWCGAHDFNFJ-RUCXOUQFSA-N (2s)-2,5-bis(azanyl)-5-oxidanylidene-pentanoic acid Chemical compound OC(=O)[C@@H](N)CCC(N)=O.OC(=O)[C@@H](N)CCC(N)=O NWNOWCGAHDFNFJ-RUCXOUQFSA-N 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 206010025421 Macule Diseases 0.000 description 1
- 201000007737 Retinal degeneration Diseases 0.000 description 1
- 208000027073 Stargardt disease Diseases 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000004258 retinal degeneration Effects 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
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Abstract
The invention relates to a human retinal pigment epithelial cell separation and cryopreservation method which includes the steps: firstly, cleaning and washing retinal pigment epithelial cells by nutrient solution; secondly, mixing Dispase II separase and DPBS buffer solution according to the ratio of 1:(40-50)w/v to prepare Dispase II separase solution; thirdly, mixing the Dispase II separase solution and Tryple select solution according to the volume ratio of 1:(2-3) to prepare digestive fluid for digestive separation; finally, performing cryopreservation after counting and centrifuging. The separation method is simple, the steps are easily controlled, the digestive fluid prepared by mixing the Dispase II separase solution with the Tryple select solution is used for performing digestive separation for the retinal pigment epithelial cells, adherence removing time of the retinal pigment epithelial cells can be shortened, cell integrity is kept, and cell injury is greatly reduced.
Description
Technical field
The present invention relates to a kind of separation of human retinal pigment epithelial cells, cryopreservation methods, belong to biomaterials art
Field.
Background technology
Retinal degeneration is the Etiological causing irreversible diseases causing blindness, wherein mainly includes age related
Degeneration of macula, retinitis pigmentosa, Stargardt disease.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 China are about super at present
Cross 20,000,000 people, and be expected to double in the year two thousand fifty.Therefore study human retinal pigment epithelial cells and be found for
Property therapeutic scheme be one of emphasis problem of current biologic medical direction research.Disappearing in human retinal pigment epithelial cells
Traditional pancreatin effect on driving birds is not good during change：General to retinal pigment epithelium digestion power, release adherent ability relatively
Weaker, retinal pigment epithelium is not released adhered state by can completely；Releasing to retinal pigment epithelium
The adherent time can not determine completely, needs to carry out groping just to can determine that；Can destroying retinal pigment epithelium as extended digestion time
The function of cell.The retinal pigment epithelium digestion method that currently there are digests for pancreatin, and its working method is：
(1) retinal pigment epithelium is taken out from CO2 gas incubator, suck supernatant.
(2) clean 1 time with DPBS (CTS) 5ml.
(3) add pancreatin 2ml, put into digestion 15min in 37.0 DEG C of CO2 gas incubator.
(4) time terminate after by cell be transferred in the culture medium containing serum terminate digestion.
Content of the invention
The invention aims to solving the problems, such as that existing digestive enzyme is not suitable for processing retinal pigment epithelium,
Provide a kind of separation of human retinal pigment epithelial cells, cryopreservation methods.
The present invention adopts the following technical scheme that：A kind of separation of human retinal pigment epithelial cells, cryopreservation methods, including
Following steps：
A kind of separation of human retinal pigment epithelial cells, cryopreservation methods it is characterised in that：Comprise the steps：
(1) remove culture fluid：Retinal pigment epithelium is taken out from CO2 gas incubator, sucks culture fluid；
(2) wash：Add DPBS (CTS) buffer 5～10ml into culture bottle, gently shake up, remove after washing completely
Liquid；
(3) digest：Dispase II is separated enzyme and DPBS buffer presses 1:40-50w/v mixes, and makes II point of Dispase
From enzymatic solution, it is 1 by volume that Dispase II separates enzymatic solution and Tryple select solution:2-3 is mixed and made into Digestive system,
Add Digestive system 2～3ml in the culture bottle in step (2), put into after fully mixing and in CO2 gas incubator, continue culture
10～15min；
(4) prepare：Add retinal pigment epithelium culture medium 3～5ml standby in centrifuge tube；
(5) terminate：Retinal pigment epithelium is taken out from CO2 gas incubator, adds 2～3ml retinal color
Plain epithelial cell culture medium terminating reaction, blows and beats cell, retinal pigment epithelium is collected to ready centrifuge tube；
(6) count：50～100ul cell suspension is taken to carry out Cell viability meter after retinal pigment epithelium is mixed
Number；
(7) it is centrifuged：By the retinal pigment epithelium gathering with 1800～2200rpm/min be centrifuged, centrifugation time be 5～
10min；
(8) frozen：Centrifugation terminates rear supernatant discarded, adds frozen stock solution Cryo-SFM according to cell counts, and adjustment is thin
Born of the same parents' density is 1 × 106/ ml, carries out frozen in subpackage to cryopreservation tube.
Further, adopt DPBS (CTS) buffer solution 3-5 time in described step (2).
Further, condition of culture in CO2 gas incubator for the described retinal pigment epithelium is temperature 37-
37.5 DEG C, 5-5.5% gas concentration lwevel.
Further, comprise group in described retinal pigment epithelium culture medium to be divided into：α MEM culture medium, N1 add
Agent, L-Glutamine, nonessential amino acid, taurine, hydrocortisone, trilute and human blood platelets lysate.
Further, preserve 30-35min when frozen in described step (8) first under the conditions of 3.5-4 DEG C, then-
It is placed in -80-85 DEG C of preservation 8-8.5h after 20--25 DEG C of preservation 2-2.5h, be finally transferred in liquid nitrogen container.
Separation method of the present invention is simple, and step is easily controllable, separates enzyme using Dispase II and Tryple select is molten
Liquid is mixed and made into Digestive system and retinal pigment epithelium is carried out with digestion separation, can shorten retinal pigment epithelium solution
Except the adherent time, keep the integrity of cell, the damage to cell substantially reduces simultaneously.
Brief description
Fig. 1 is remaining result schematic diagram in culture bottle after the embodiment of the present invention 1 Digestive system digestion 15min.
Fig. 2 is remaining result schematic diagram in culture bottle after comparative example 1 pancreatin digestion 15min of the present invention.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.
L-Glutamine in the present invention, nonessential amino acid are purchased from LIFE TECHOLOGISE company；DPBS (CTS) is purchased from
GIBCO company；Dispase II is purchased from ROCHE Roche Holding Ag；Pancreatin is purchased from INVITROGEN company；α MEM culture medium, N1 add
Agent, taurine, hydrocortisone, trilute are purchased from SIGMA company；It is public that human blood platelets lysate is purchased from HELIOS
Department；Cryo-SFM is purchased from PromoCell company
The culture medium of retinal pigment epithelium is prepared：α modifies lower bound minimal medium (MEM, α modification)
500ml, N1 additive (N1supplement) 5ml, L-Glutamine (Glutamine) 5ml, nonessential amino acid
(Nonessentialaminoacids) 5ml, taurine (Taurine) 150mg, hydrocortisone (Hydrocortisone)
15ug, trilute (Triiodo-thyronin) 0.010ug, human blood platelets lysate (HPL) 50ml.
Embodiment 1：
A kind of separation of human retinal pigment epithelial cells, cryopreservation methods, comprise the steps：
(1) remove culture fluid：Retinal pigment epithelium is taken out from CO2 gas incubator, sucks culture fluid.
(2) wash：Add DPBS (CTS) 5ml into culture bottle, gently shake up, after washing completely, remove liquid.
(3) digest：Add retinal pigment epithelium Digestive system 2ml in culture bottle, after fully mixing, put into dioxy
Change 10min in carbon incubator.
(4) prepare：Take 15ml centrifuge tube 1, add retinal pigment epithelium culture medium 3ml standby；
(5) terminate：Retinal pigment epithelium is taken out after terminating by the time, adds 2ml retinal pigment epithelium
Culture medium terminating reaction, is carried out with Manual liquid transfering device blowing and beating cell, cell is collected to ready 15ml centrifuge tube；
(6) observe：Discarded culture bottle is placed under inverted microscope and observes the remaining situation of digestion；
(7) count：50ul cell suspension is taken to carry out Cell viability counting after cell is mixed；
(8) it is centrifuged：By the cell gathering with 1800rpm/min, 5min is centrifuged；
(9) frozen：Centrifugation terminates rear supernatant discarded, adds appropriate frozen stock solution according to cell counts, and adjustment cell is close
Spend for 1 × 106/ ml, in subpackage to 2ml cryopreservation tube, often pipe 500ul.
The process for preparation of Digestive system：The DPBS buffer of the Dispase II of 1g, 50mL, the Tryple select of 100mL,
After fully dissolving mixes, subpackage is placed on -20 DEG C of preservations
Embodiment 2：
A kind of separation of human retinal pigment epithelial cells, cryopreservation methods, comprise the steps：
(1) remove culture fluid：Retinal pigment epithelium is taken out from CO2 gas incubator, sucks culture fluid；
(2) wash：Add DPBS (CTS) 10ml into culture bottle, gently shake up, after washing completely, remove liquid；
(3) digest：Add retinal pigment epithelium Digestive system 3ml in culture bottle, after fully mixing, put into dioxy
Change 15min in carbon incubator；
(4) prepare：Take 15ml centrifuge tube 1, add retinal pigment epithelium culture medium 5ml standby；
(5) terminate：Retinal pigment epithelium is taken out after terminating by the time, adds 3ml retinal pigment epithelium
Culture medium terminating reaction, is carried out with Manual liquid transfering device blowing and beating cell, cell is collected to ready 15ml centrifuge tube；
(6) observe：Discarded culture bottle is placed under inverted microscope and observes the remaining situation of digestion；
(7) count：100ul cell suspension is taken to carry out Cell viability counting after cell is mixed；
(8) it is centrifuged：By the cell gathering with 2200rpm/min, 10min is centrifuged；
(9) frozen：Centrifugation terminates rear supernatant discarded, adds appropriate frozen stock solution according to cell counts, and adjustment cell is close
Spend for 1 × 106/ ml, in subpackage to 2ml cryopreservation tube, often pipe 500ul.
The process for preparation of Digestive system：The DPBS buffer of the Dispase II of 1g, 40mL, the Tryple select of 120mL,
After fully dissolving mixes, subpackage is placed on -20 DEG C of preservations
Comparative example 1：
Concretely comprise the following steps：
(1) remove culture fluid：Retinal pigment epithelium is taken out from CO2 gas incubator, sucks culture fluid；
(2) wash：Add DPBS (CTS) 5ml into culture bottle, gently shake up, after washing completely, remove liquid；
(3) digest：Add pancreatin 2ml, after fully mixing, put into 15min in CO2 gas incubator；
(4) prepare：Take 15ml centrifuge tube 1, add retinal pigment epithelium culture medium 3ml standby；
(5) terminate：Cell is taken out after terminating by the time, adds 2ml retinal pigment epithelium culture medium terminating reaction,
Carried out with Manual liquid transfering device blowing and beating cell, cell is collected to ready 15ml centrifuge tube；
(6) observe：Discarded culture bottle is placed under inverted microscope and observes the remaining situation of digestion；
(7) count：50ul cell suspension is taken to carry out Cell viability counting after cell is mixed；
(8) it is centrifuged：By the cell gathering with 2000rpm/min, 5min is centrifuged；
(9) frozen：Centrifugation terminates rear supernatant discarded, adds appropriate frozen stock solution according to cell counts, and adjustment cell is close
Spend for 1 × 106/ ml, in subpackage to 2ml cryopreservation tube, often pipe 500ul.
Respectively the cell in embodiment 1 and comparative example 1 is carried out with Cell viability counting, every part of sample takes six parts, and obtains
Average, result is as shown in table 1.
Table 1
As shown in Table 1, the Cell viability average of retinal pigment epithelium Digestive system digestion is adopted to exist in embodiment 1
More than 95.0%.
It is less than 92.0% with the Cell viability average of pancreatin digestion, less than retinal pigment epithelium in comparative example 1
The cell that Digestive system is processed.
Observe the remaining situation of digestion with the Tissue Culture Flask of Digestive system digestion as shown in figure 1, under identical digestion condition,
Digestive system digests used time 10min, and cell dissociation effect is good, and in the visual field, visible attached cell quantity is little.
Observe the remaining situation of digestion with the Tissue Culture Flask of pancreatin digestion as shown in Fig. 2 under identical digestion condition, pancreas
Enzymic digestion used time 20min, cell dissociation effect is poor, and in the visual field, visible attached cell quantity is very many.
Claims (5)
1. a kind of separation of human retinal pigment epithelial cells, cryopreservation methods it is characterised in that：Comprise the steps：
（1）Remove culture fluid：Retinal pigment epithelium is taken out from CO2 gas incubator, sucks culture fluid；
（2）Washing：DPBS is added into culture bottle（CTS）Buffer 5 ~ 10ml, gently shakes up, and removes liquid after washing completely；
（3）Digestion：Dispase II is separated enzyme and DPBS buffer presses 1:40-50w/v mixes, and makes Dispase II and separates enzyme
Solution, it is 1 by volume that Dispase II separates enzymatic solution and Tryple select solution:2-3 is mixed and made into Digestive system, Xiang Bu
Suddenly（2）In culture bottle in add Digestive system 2 ~ 3ml, put into after fully mixing continue in CO2 gas incubator culture 10 ~
15min；
（4）Prepare：Add retinal pigment epithelium culture medium 3 ~ 5ml standby in centrifuge tube；
（5）Terminate：Retinal pigment epithelium is taken out from CO2 gas incubator, adds on 2 ~ 3ml retinal pigment
Chrotoplast culture medium terminating reaction, blows and beats cell, retinal pigment epithelium is collected to ready centrifuge tube；
（6）Count：50 ~ 100ul cell suspension is taken to carry out Cell viability counting after retinal pigment epithelium is mixed；
（7）Centrifugation：The retinal pigment epithelium gathering is centrifuged with 1800 ~ 2200 rpm/min, centrifugation time be 5 ~
10min；
（8）Frozen：Centrifugation terminates rear supernatant discarded, adds frozen stock solution Cryo-SFM according to cell counts, and adjustment cell is close
Spend for 1 × 106/ ml, carries out frozen in subpackage to cryopreservation tube.
2. the separation of human retinal pigment epithelial cells as claimed in claim 1, cryopreservation methods it is characterised in that：Described
Step（2）Middle employing DPBS（CTS）Buffer solution 3-5 time.
3. the separation of human retinal pigment epithelial cells as claimed in claim 1, cryopreservation methods it is characterised in that：Described
Condition of culture in CO2 gas incubator for the retinal pigment epithelium is temperature 37-37.5 DEG C, 5-5.5% carbon dioxide
Concentration.
4. the separation of human retinal pigment epithelial cells as claimed in claim 1, cryopreservation methods it is characterised in that：Described
Comprise group in retinal pigment epithelium culture medium to be divided into：α MEM culture medium, N1 additive, L-Glutamine, nonessential amino
Acid, taurine, hydrocortisone, trilute and human blood platelets lysate.
5. the separation of human retinal pigment epithelial cells as claimed in claim 1, cryopreservation methods it is characterised in that：Described
Step（8）In frozen when first under the conditions of 3.5-4 DEG C preserve 30-35min, then -20-25 DEG C preserve 2-2.5h rearmounted
Preserve 8-8.5h in -80-85 DEG C, be finally transferred in liquid nitrogen container.
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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 |
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