CN102763639B - Composite anti-freezing liquid and application thereof, and method for preserving human amnion by using same - Google Patents
Composite anti-freezing liquid and application thereof, and method for preserving human amnion by using same Download PDFInfo
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Abstract
The invention discloses a composite anti-freezing liquid, and a method for preserving human amnion by using the same. The composite anti-freezing liquid comprises a basic anti-freezing liquid composed of DMEM culture medium, glycerol and dimethyl sulfoxide at volume ratio of (4.5-5.5):(3.5-4.5):(0.5-1.5); and trehalose having a concentration of 0.05-0.15 mol/L in the composite anti-freezing liquid. After pretreatment by the composite anti-freezing liquid, fresh human amnion can be directly preserved in liquid nitrogen without refrigerating with programmed cooling instrument, so as to simplify procedure, save cost, shorten pretreatment time, reduce pollution, and maintain amnion activity in long term.
Description
Technical field
The present invention relates to histocyte Techniques of preserving field, be specifically related to a kind of compound anti frozen liquid and the application in people's amnion liquid nitrogen is preserved thereof and use the method for this anti frozen liquid depositary amnion.
Background technology
People's amnion (hereinafter to be referred as amnion) is divided into epithelial layer, basement membrane layer, compacted zone, fibroblast layer and spongy layer, and wherein latter three layers are called again hypothallus.In amnion, there are cell and matrix, wherein cell is divided into again amniotic epithelial cells (for stem cell, be distributed in epithelial layer) and amnion mesenchymal stem cell (being distributed in hypothallus), two kinds of stem cells of this of amnion have been proved to be the stem cell characteristic all having further to different tissues Cell Differentiation and secretion cytokine profiles.Its matrix is also good cytoskeleton material.In addition, amnion also have antigenicity weak, without oncogenicity, be not subject to the advantage of source and ethics restriction.Therefore, amnion is more and more extensive in the application in a plurality of fields of medical science, demand is also increasing.
Solve amnion and be by fresh amnion long preservation and set up amnion storehouse in the measure of the increasing demand in a plurality of fields, and make amnion cell and the matrix of preserving active similar to fresh amnion, once need at any time preservation amnion rewarming can be applied.
The preservation of amnion is at present divided into normal temperature and profound hypothermia is preserved two large classes: 1, normal temperature store method is simple, exactly fresh amnion is placed on and in some culture fluid, is placed in general refrigerator and preserves, but the holding time is short, its active basic forfeiture after 1～2 week, and the method obviously can only be carried out of short duration preservation to fresh amnion.2, profound hypothermia store method is divided into again-80 ℃ of low temperature refrigerators and preserves with-196 ℃ of liquid nitrogen and preserve, because the latter compared with the long preservation of the former longer (in theory can " forever " preservation) for amnion holding time.
External Lee and Tseng adopt DMEM (Dulbecco ' s modified Eagle ' s medium)/glycerine by antifreeze, successfully amnion to be stored to ，Ci Fabei FDA (Food and Drug Adminstration) under-80 ℃ of conditions and recommended; But also there are some researches show that the amnion activity that DMEM/ glycerine profound hypothermia is preserved falls sharply, its reason may be that completely not suppressed, cellular metabolism (the comprising enzymes metabolism) process of molecular motion does not stop completely under-80 ℃ of conditions, along with the prolongation cytoactive of holding time lowers and even therefore function dead, amnion cell secrete cytokines is also affected or obstacle.And under-196 ℃ of liquid nitrogen conditions, various metabolism close to " hibernation " or stop, can " forever " be preserved amnion cell activity in theory in cell.
At present the domestic scholar of having adopt simplify a step preservation method (take (M199 liquid/lO%DMSO is antifreeze) amnion is stored in liquid nitrogen, the method belongs to slow falling temperature method: be to put into liquid nitrogen preservation after amnion first being used after antifreeze pretreatment programmed cooling instrument (during with liquid nitrogen cooling) amnion is progressively dropped to-80 ℃ again.Slow falling temperature method needs expensive programmed cooling instrument, and many, the consuming time length of its operating procedure, contaminated link and expense also increase, and in the time of especially will preserving in a large number amnion and set up amnion storehouse, the deficiency of simplifying a step preservation method is apparent.If study a kind ofly without programmed cooling instrument, fast cooling method that preservation effect is good, that is: will directly put into liquid nitrogen through the pretreated amnion of antifreeze and preserve, its advantage is self-evident.
Yet when liquid nitrogen is preserved amnion, two stages of freezing cooling and rewarming all can be caused major injury to amnion stem cell, make amnion Stem Cell Activity and the secreting function thereof preserved be subject to obvious impact.For alleviating low temperature to amnion stem cell injuries, generally acknowledged method is first with antifreeze, amnion to be carried out to pretreatment to carry out frozen again.
It is more complicated that antifreeze alleviates in refrigerating process the mechanism of action to tissue damage, may be relevant with following factors: 1, lower ice crystal and form speed and size, thereby alleviate the coup injury of ice crystal to cell; 2, alleviate the inside and outside ice crystal of cell and formed the inside and outside permeability variation of the cell bringing; 3, the structure of stabilizing cell membrane, improves cell membrane fluidity, alleviates cell membrane damage; 4, dimethyl sulfoxide (DMSO) (DMSO) also has oxygen radical removing effect.But antifreeze (especially high concentration penetrability antifreeze) itself also can cause cellular damage.In prior art, antifreeze is divided into two kinds of penetrability and non-penetratives, conventional antifreeze has dimethyl sulfoxide (DMSO) (DMSO), propane diols, glycerine etc., and various antifreeze mechanism of action are different.The requirement and the toxicity that due to single antifreeze, are difficult to reach cryoprotection are also large, and therefore the normal antifreeze that adopts multiple different mechanism of action constitutes jointly the protectant of Cryopreserved at present.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of compound anti frozen liquid and application and this anti frozen liquid of use depositary amnion.Adopting compound anti frozen liquid of the present invention to carry out not needing the cooling of service routine cooling instrument to be directly placed in liquid nitrogen after pretreatment to Freshman amnion preserves, simplify step, shortened the pretreated time in cost-saving, reducing pollution, and can permanently effectively maintain amnion activity.
A kind of compound anti frozen liquid of the present invention, it is comprised of basic anti frozen liquid and trehalose, described basic anti frozen liquid is comprised of by the volume ratio of 4.5～5.5:3.5～4.5:0.5～1.5 DMEM culture fluid, glycerine and dimethyl sulfoxide (DMSO), and the concentration of described trehalose in compound anti frozen liquid is 0.05～0.15mol/L.
In above-mentioned compound anti frozen liquid formula,
Described basic anti frozen liquid is preferably comprised of by the preferred volume ratio of 5:4:1 DMEM culture fluid, glycerine and dimethyl sulfoxide (DMSO).Wherein DMEM culture fluid can be high glycoform can be also low-sugar type, preferred low-sugar type.
The concentration of described trehalose in compound anti frozen liquid is preferably 0.1mol/L.
Above-mentioned compound anti frozen liquid only need to measure respective components by proportional quantity and mix, be specially: first by the volume ratio of 4.5～5.5:3.5～4.5:0.5～1.5, measure DMEM culture fluid, glycerine and dimethyl sulfoxide (DMSO), mix, obtain basic anti frozen liquid, and then add trehalose to basic anti frozen liquid, controlling the concentration of trehalose in compound anti frozen liquid is 0.05～0.15mol/L.
The present invention also comprises the application of above-mentioned compound anti frozen liquid in preserving amnion activity.
The present invention also comprises the method with above-mentioned compound anti frozen liquid depositary amnion, specifically people's amnion is dipped in to 3～10min in 0～4 ℃ of compound anti frozen liquid, take out, be laid between two-layer aseptic plastic film and seal with sealing machine, one deck plastic tube tiles on plastic film after sealing, plastic foil and plastic tube after sealing are rolled into tubular, and overcoat rubber band is fixed, and after numbering, inserts in liquid nitrogen container and preserves immediately.By one deck plastic tube that tiles on the plastic film after sealing, be rolled into tubular, the inside and outside layer that makes to include the plastic film of amnion when freezing is caught a cold evenly again.
Adopt the Thawing Methods of people's amnion of said method preservation to be: to take out people's amnion rewarming 90～120s in 40～42 ℃ of stroke-physiological saline solution water-baths, then put into 0～4 ℃ of stroke-physiological saline solution and remove the compound anti frozen liquid on people's amnion.
Compared with prior art, feature of the present invention is:
1, described anti frozen liquid is selected penetrability antifreeze (glycerine, dimethyl sulfoxide (DMSO)) and non-penetrative antifreeze (trehalose) combination, the former enters cell interior by rapid permeability, electrolyte in dilution born of the same parents, reduce the solution effect in refrigerating process, change the arrangement of hydrone, thereby change the inside and outside ice crystal structure of born of the same parents; Thereby trehalose makes cell dehydration before freezing, avoid antifreezing agent to infiltrate excessive expansion and permeability damage that cell causes cell, thereby reach the object that reduces structure of biological macromolecule in film phase transition temperature, stabilized cell and reach good vitrifying characteristic.
2, with above-mentioned compound anti frozen liquid, be directly placed in fast the method for liquid nitrogen after to fresh amnion pretreatment, mainly to be by adhesion and the substrate composed very thin semi-transparent film with half permeability based on amnion, good to antifreeze permeability, after fast cooling, can make antifreeze form a kind of have full-bodied between liquid and solid-state between, non-crystal state, rambling, transparent vitreousness, the inside and outside liquid of born of the same parents can pass through rapidly the freezing sensitizing range of-5～-15 ℃, avoided the ice crystal in frozen process to form, reduced the damage to amniotic epithelial cells, in addition, under-196 ℃ of conditions, molecular motion is suppressed completely, and the reaction of the whole physiology and chemistry of cell almost completely stops, thereby cell is in resting state, thereby can preserve long-term effectively amnion activity.
3, do not need after using compound anti frozen liquid of the present invention to process fresh amnion the cooling of service routine cooling instrument to be directly placed in liquid nitrogen and preserve, simple to operate, cost is low, reduced opportunities for contamination.
Accompanying drawing explanation
Fig. 1 carries out the cellular morphology (light microscope * 400) of the rear amnion of HE dyeing without the fresh amnion of any processing in experimental example of the present invention;
Fig. 2 is that in experimental example of the present invention, A group amnion is preserved the cellular morphology (light microscope * 400) of carrying out the rear amnion of HE dyeing after 1 month;
Fig. 3 is that in experimental example of the present invention, A group amnion is preserved the cellular morphology (light microscope * 400) of carrying out the rear amnion of HE dyeing after 3 months;
Fig. 4 is that in experimental example of the present invention, B group amnion is preserved the cellular morphology (light microscope * 400) of carrying out the rear amnion of HE dyeing after 1 month;
Fig. 5 is that in experimental example of the present invention, B group amnion is preserved the cellular morphology (light microscope * 400) of carrying out the rear amnion of HE dyeing after 3 months;
Fig. 6 is that in experimental example of the present invention, C group amnion is preserved the cellular morphology (light microscope * 400) of carrying out the rear amnion of HE dyeing after 1 month;
Fig. 7 is that in experimental example of the present invention, C group amnion is preserved the cellular morphology (light microscope * 400) of carrying out the rear amnion of HE dyeing after 3 months;
Fig. 8 is that in experimental example of the present invention, D group amnion is preserved the cellular morphology (light microscope * 400) of carrying out the rear amnion of HE dyeing after 1 month;
Fig. 9 is that in experimental example of the present invention, D group amnion is preserved the cellular morphology (light microscope * 400) of carrying out the rear amnion of HE dyeing after 3 months;
Figure 10 carries out the survival condition (light microscope * 200) of amniotic epithelial cells after Trypan Blue without the fresh amnion of any processing in experimental example of the present invention;
Figure 11 is that in experimental example of the present invention, A group amnion is preserved the survival condition (light microscope * 200) of carrying out amniotic epithelial cells after Trypan Blue after 1 month;
Figure 12 is that in experimental example of the present invention, A group amnion is preserved the survival condition (light microscope * 200) of carrying out amniotic epithelial cells after Trypan Blue after 3 months;
Figure 13 is that in experimental example of the present invention, B group amnion is preserved the survival condition (light microscope * 20) of carrying out amniotic epithelial cells after Trypan Blue after 1 month;
Figure 14 is that in experimental example of the present invention, B group amnion is preserved the survival condition (light microscope * 200) of carrying out amniotic epithelial cells after Trypan Blue after 3 months;
Figure 15 is that in experimental example of the present invention, C group amnion is preserved the survival condition (light microscope * 200) of carrying out amniotic epithelial cells after Trypan Blue after 1 month;
Figure 16 is that in experimental example of the present invention, C group amnion is preserved the survival condition (light microscope * 200) of carrying out amniotic epithelial cells after Trypan Blue after 3 months;
Figure 17 is that in experimental example of the present invention, D group amnion is preserved the survival condition (light microscope * 200) of carrying out amniotic epithelial cells after Trypan Blue after 1 month;
Figure 18 is that in experimental example of the present invention, D group amnion is preserved the survival condition (light microscope * 200) of carrying out amniotic epithelial cells after Trypan Blue after 3 months.
Embodiment
With specific embodiment, the invention will be further described below, but the present invention is not limited to these embodiment.
Embodiment 1: compound anti frozen liquid
By the volume ratio of 5:4:1, measure DMEM culture fluid, glycerine and dimethyl sulfoxide (DMSO), mix, obtain basic anti frozen liquid, and then add trehalose in basic anti frozen liquid, controlling the concentration of trehalose in compound anti frozen liquid is 0.1mol/L, and obtaining trehalose final concentration is the compound anti frozen liquid of 0.1mol/L.
Embodiment 2: compound anti frozen liquid
By the volume ratio of 4.5:4.5:1, measure DMEM culture fluid, glycerine and dimethyl sulfoxide (DMSO), mix, obtain basic anti frozen liquid, and then add trehalose in basic anti frozen liquid, controlling the concentration of trehalose in compound anti frozen liquid is 0.1mol/L, and obtaining trehalose final concentration is the compound anti frozen liquid of 0.1mol/L.
Embodiment 3: compound anti frozen liquid
By the volume ratio of 5.5:3.5:1.5, measure DMEM culture fluid, glycerine and dimethyl sulfoxide (DMSO), mix, obtain basic anti frozen liquid, and then add trehalose in basic anti frozen liquid, controlling the concentration of trehalose in compound anti frozen liquid is 0.05mol/L, and obtaining trehalose final concentration is the compound anti frozen liquid of 0.05mol/L.
Embodiment 4: compound anti frozen liquid
By the volume ratio of 5:3.5:0.5, measure DMEM culture fluid, glycerine and dimethyl sulfoxide (DMSO), mix, obtain basic anti frozen liquid, and then add trehalose in basic anti frozen liquid, controlling the concentration of trehalose in compound anti frozen liquid is 0.15mol/L, and obtaining trehalose final concentration is the compound anti frozen liquid of 0.15mol/L.
Experimental example: the experiment of preserving amnion
1, experiment material:
Guilin hospital cuts open palace and produces 12, Freshman placenta, is full-term pregnancy pregnant woman, and age 25～3O year obtaining the placenta time is postoperative 0.5～1h.Antenatal by Serological testing pregnant woman without human immunodeficiency virus, B-mode, hepatitis C virus and syphilis.Placenta is rinsed to remove hematocele repeatedly by the stroke-physiological saline solution that contains 50 μ g/ml penicillin, 50 μ g/ml streptomycins, 100 μ g/ml neomycins and 2.5 μ g/ml amphomoronals, the chorion that epithelial surface hematocele and basal surface are remaining and vascular tissue, blunt separation is also cut complete amnion and is put in an aseptic square position, now visually observes it and is half transparent milk white film.Then to be cut into 5.0cm * 5.0cm size square block standby with aseptic.
2, experiment anti frozen liquid
Compound anti frozen liquid 1: get 8ml DMEM culture fluid (low-sugar type, Sai Mo flies generation Lovell biochemistry goods (Beijing) Co., Ltd) and 8ml glycerine (DMEM: glycerine=1:1, volume) and mix, obtain.
Compound anti frozen liquid 2: get 10ml DMEM culture fluid (low-sugar type, Sai Mo flies generation Lovell biochemistry goods (Beijing) Co., Ltd), 8ml glycerine and 2ml dimethyl sulfoxide (DMSO) (DMEM: glycerine: dimethyl sulfoxide (DMSO)=5:4:1, volume) mix, obtain.
Compound anti frozen liquid 3: get 10ml DMEM culture fluid (low-sugar type, Sai Mo flies generation Lovell biochemistry goods (Beijing) Co., Ltd), 8ml glycerine and 2ml dimethyl sulfoxide (DMSO) (DMEM: glycerine: dimethyl sulfoxide (DMSO)=5:4:1, volume) mix, obtain basic anti frozen liquid, and then to basic anti frozen liquid, add the trehalose of 0.684g, obtaining trehalose final concentration is the compound anti frozen liquid of 0.1mol/L.
3, experiment grouping and store method
The above-mentioned fresh amnion of obtaining is divided into A group, B group, C group and D group, wherein:
A group as a control group, be immersed in 5min in 0～4 ℃ of physiological saline, take out, be laid between two-layer aseptic plastic film and seal with sealing machine, one deck plastic tube tiles on plastic film after sealing, plastic foil and plastic tube after sealing are rolled into tubular, and overcoat rubber band is fixed, and after numbering, inserts in liquid nitrogen container and preserves immediately.
B group, C group and D group be as experimental group, B group, C group and D group carried out to be directly placed in liquid nitrogen after pretreatment with above-mentioned compound anti frozen liquid 1, compound anti frozen liquid 2 and compound anti frozen liquid 3 respectively and preserve, and concrete grammar is:
B group, C group and D group amnion are immersed in respectively to 5min in 0～4 ℃ of physiological saline, take out, be laid in respectively between two-layer aseptic plastic film and seal with sealing machine, respectively at one deck plastic tube that tiles on the plastic film after sealing, plastic foil and plastic tube after sealing are rolled into tubular, overcoat rubber band is fixed, and after numbering, inserts in liquid nitrogen container and preserves immediately.
4, Thawing Methods
Each group amnion is placed in rapidly to 40～42 ℃ of stroke-physiological saline solution rewarmings 100 seconds from liquid nitrogen takes out, then each group amnion is put into respectively to 4 ℃ of stroke-physiological saline solution and remove the compound anti frozen liquid on amnion.
5, amnion cell structure, activity and secreting function detection method and result
5.1HE colouring method and testing result
Getting above-mentioned fresh amnion (without any processing) carries out HE dyeing (HE colouring method is with reference to pathological examination technology, Deng Buhua, Higher Education Publishing House, 2005 the 1st edition: 80-83, lower with), and amnion cell form is checked to (under light microscope * 400), as shown in Figure 1, visible, epithelial layer is comprised of simple cuboidal epithelium cell, and cellular morphology is intact, and karyon is clear placed in the middle, marshalling is tight, and its lower visible one deck is continuous, the red basilar memebrane dying of homogeneous.
Above-mentioned A group, B group, C group and D group are preserved after 1 month and 3 months by method described in above-mentioned 3, sample respectively, after sample rewarming, carry out respectively HE dyeing, and the structure of each group amnion is compared to (under light microscope * 400), its result is as follows:
A group: preserve visible epithelial cell distortion in 1 month, not of uniform size, iuntercellular is apart from increase, the shrinkage of karyon engrain, cell membrane is more clear, and basilar memebrane is slightly complete, specifically as shown in Figure 2; Preserve visible epithelial cell distortion in 3 months serious, arrange sparse, part and come off, karyon is irregular, part hypochromatosis, and cell membrane is owed clearly, cytomixis, basilar memebrane is smudgy, specifically as shown in Figure 3.
B group: preserve 1 month visible epithelial cell fasciation, arrange closelyr, cell nucleus is shrinkage slightly, and basilar memebrane is more complete, specifically as shown in Figure 4; Preserve visible epithelial cell distortion in 3 months, arrange compactness, karyon pyknosis, the close cell based of part bottom, part cytomixis, basilar memebrane attenuation and discontinuous, specifically as shown in Figure 5.
C group: preserve 1 month visible epithelial cell structural integrity, queueing discipline, basilar memebrane is continuous, specifically as shown in Figure 6; Preserve 3 months visible cells and karyon flattening, iuntercellular is apart from increasing, and basilar memebrane is more complete, specifically as shown in Figure 7.
D group: it is intact to preserve 1 month visible epithelial cell structure, and karyon form is normal, cell arrangement is compact, and basilar memebrane is preserved complete, and morphosis more approaches fresh amnion, specifically as shown in Figure 8; Preserve 3 months cell structures with preserve 1 month close, specifically as shown in Figure 9.
5.2 Trypan Blue
To the above-mentioned fresh amnion of obtaining (without any processing) carry out Trypan Blue (Trypan Blue be with reference to Xu Liying, Chen Jiaqi, Zhou Shiyou etc., the activity of fresh amnion maintains method research, Chinese Journal of Practical Ophthalmology, 2002,2 (20): 137-139., lower same), its survival condition as shown in figure 10, visible epithelial cell is all survived, and cell structure is clear, be evenly distributed (under light microscope * 200).
Above-mentioned A group, B group, C group and D group are preserved after 1 month and 3 months by method described in above-mentioned 3, sample respectively, after sample rewarming, carry out respectively Trypan Blue, and the survival condition of each group amnion is compared to (under light microscope * 200), its result is as follows:
A group: preserve 1 month visible epithelial cell most dead (indigo plant is dyed), cell structure is clear, specifically as shown in figure 11; The epithelial cell of preserving 3 months visible most amnions is dead, part clasmatosis, specifically as shown in figure 12.
B group: preserve 1 month visible amniotic epithelial cells Mortality, cell structure is clear, is evenly distributed, specifically as shown in figure 13; Preserve 3 months visible epithelial cell Large Scale Deaths, part cell clear in structure, part clasmatosis, specifically as shown in figure 14.
C group: preserve the most of survival of visible amniotic epithelial cells in 1 month, a small amount of cell death, cell structure is clear, is evenly distributed, specifically as shown in figure 15; Preserve 3 months dead quantity of visible epithelial cell and increase, cell structure is clear, a small amount of clasmatosis, specifically as shown in figure 16.
D group: preserve visible most amniotic epithelial cells survivals in 1 month, cell structure is clear, is evenly distributed, specifically as shown in figure 17; Preserve amniotic epithelial cells survival in 3 months good, 1 month result is close with preserving, specifically as shown in figure 18.
Improvement method with reference to Trypan Blue exclusive method, amnion piece is laid in to (epithelial surface upwards) on culture dish, and PBS liquid rinsing twice, directly adds 0.1% to expect blue dyestuff, under room temperature, (20～22 ℃) are contaminated 3 minutes, take out amnion piece and again with PBS liquid, rinse 1min.Amnion is laid on slide, and under light microscope, (* 200) are calculated epithelial cell and are dyed to blue dead cell percentage, each random 6 regions of selecting, and 1000 cells are calculated in each region altogether.The active testing result of amnion cell as described in Table 1.
Table 1: each group is preserved amniotic epithelial cells lethality measurement result
D group compares with each group, * * P < 0.01; * P < 0.05.
From table 1 data, the amniotic epithelial cells survival rate of D prescription method preservation is the highest, preservation effect is best.
5.3 immunohistochemistries detect and result
Get and respectively organize amnion piece and fix with 4% paraformaldehyde, serial section after paraffin embedding, thickness approximately 4 μ m.EGF immunohistochemical staining paraffin section enzymic digestion program is as follows: paraffin section de-waxing is to water, the fresh configuration of distilled water 3%H
2o
2, room temperature 10 minutes, with deactivating endogenous peroxydase.Distillation washing 2 minutes * 3 times.Drip compound digestive juice, put 37 ℃ of water baths of people interior 20 minutes, distillation washing 2 minutes * 3 times.Drip lowlenthal serum confining liquid, room temperature 20 minutes, sealing nonspecific binding site.Get rid of unnecessary liquid, do not wash.Drip EGF-anti-(rabbit igg), in 37 ℃ of water baths, hatch 1.5 hours, 0.01M PBS washes 2 minutes * 3 times.PBS replaces 1 anti-dyeing as negative control.Drip the anti-rabbit igg of biotinylated goat, in 37 ℃ of water baths 20 minutes, 0.01M PBS washed 2 minutes * 3 times, drips reagent SABC, 37 ℃ 20 minutes.0.01M PBS washes 10 minutes * 4 times.DAB colour developing, controls the reaction time under mirror, approximately 5 minutes.Distilled water washing 5～7 minutes.Dehydration, transparent, mounting.Microscopic examination, photograph, 6 high power fields of every group of random selection, and application image analytical system (Image-Pro Plus 6.0) is measured the average integral optical density value of amnion epithelial layer.The expression (SABC) of the amnion cell factor (EGF) as described in Table 2.
In the different holding time amnion EGF expression of each group of table 2 group, compare (IOD/ μ m
2,
)
D group compares with each group, * * P < 0.01; * P < 0.05.
From table 2 data, the amniotic epithelial cells EGF that D prescription method is preserved express the highest and with the fresh amnion group (n=6,0.3588 ± 0.0283) basically identical (P > 0.05) of preserving without profound hypothermia.
From above experimental data:, EGF good with compound anti frozen liquid of the present invention the amnion structural integrity, the epithelial cell survival that adopt the method for the invention to preserve fresh amnion still has higher expression, proves by compound anti frozen liquid of the present invention and described method and can maintain preferably amnion activity.
Claims (6)
1. a compound anti frozen liquid, it is characterized in that: it is comprised of basic anti frozen liquid and trehalose, described basic anti frozen liquid is comprised of by the volume ratio of 4.5～5.5:3.5～4.5:0.5～1.5 DMEM culture fluid, glycerine and dimethyl sulfoxide (DMSO), and the concentration of described trehalose in compound anti frozen liquid is 0.05～0.15mol/L.
2. compound anti frozen liquid according to claim 1, is characterized in that: described basic anti frozen liquid is comprised of by the volume ratio of 5:4:1 DMEM culture fluid, glycerine and dimethyl sulfoxide (DMSO).
3. the compound anti frozen liquid described in claim 1 or 2, is characterized in that: the concentration of described trehalose in compound anti frozen liquid is 0.1mol/L.
4. the application of the compound anti frozen liquid described in any one in depositary's amnion activity in claim 1～3.
5. right to use requires the method for the compound anti frozen liquid depositary amnion described in any one in 1～3, it is characterized in that: be that people's amnion is dipped in to 3～10min in 0～4 ℃ of compound anti frozen liquid, take out, be laid between two-layer aseptic plastic film and seal with sealing machine, one deck plastic tube tiles on plastic film after sealing, plastic foil and plastic tube after sealing are rolled into tubular, and overcoat rubber band is fixed, and after numbering, inserts in liquid nitrogen container and preserves immediately.
6. method according to claim 5, it is characterized in that: the Thawing Methods of the people's amnion through preserving is: take out people's amnion rewarming 90～120s in 40～42 ℃ of stroke-physiological saline solution water-baths, then put into 0～4 ℃ of stroke-physiological saline solution and remove the compound anti frozen liquid on people's amnion.
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| CN113101415A (en) * | 2021-03-25 | 2021-07-13 | 北京桀亚生物医学研究有限公司 | Biological amniotic membrane with complete degradation and controllable cell structure and preparation method thereof |
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