CN106719600A - A kind of adipose tissue frozen stock solution - Google Patents
A kind of adipose tissue frozen stock solution Download PDFInfo
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- CN106719600A CN106719600A CN201611082588.5A CN201611082588A CN106719600A CN 106719600 A CN106719600 A CN 106719600A CN 201611082588 A CN201611082588 A CN 201611082588A CN 106719600 A CN106719600 A CN 106719600A
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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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- 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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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Abstract
The present invention relates to biological technical field, a kind of adipose tissue frozen stock solution is disclosed, the adipose tissue frozen stock solution has to adipose tissue and good freezes effect.Adipose tissue frozen stock solution disclosed by the invention is based on volumetric concentration including following component：Dimethyl sulfoxide (DMSO) 10~20%, basal medium 65~90% and dextran 1~20% containing nonessential amino acid, the frozen stock solution is also including trehalose that content is 0.5~1.5g/ml.The invention also discloses a kind of cryopreservation methods of adipose tissue, comprise the following steps：Pretreated adipose tissue is put into be made in the cryopreservation tube containing above-mentioned frozen stock solution and freezes sample, then the sample that freezes is put into program temperature reduction box, described program cooling box is put into 80 DEG C of refrigerators again, the sample that freezes is transferred to liquid nitrogen after 24h, complete to freeze.The invention also discloses application of the adipose tissue frozen stock solution in biological tissue's preservation.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of adipose tissue frozen stock solution.
Background technology
Adipose tissue is mainly made up of the fat cell of a large amount of clusters, assembles pockets of fat cell by the loose connective of thin layer
Tissue is separated into leaflet, and the reticular fibre in adipose tissue is very flourishing.Yellow (white) color adipose tissue is (dynamic in some lactations in yellow
Thing is white).Adipose tissue is aggregative by a large amount of unilocular adipose cells, and fat cell is rounded or polygon, cell center
There are a big fat drips, kytoplasm is in thin layer, positioned at cell periphery, wrap fat drips.In section, fat drips are dissolved into a big vacuole；Born of the same parents
Core is oblate, and cell side is pushed through by fat drips, together with part kytoplasm in crescent.Yellow adipose tissue is mainly distributed on subcutaneous
Tissue, nethike embrane and mesenterium etc., the 10%~20% of the general about percentage of liveweight of man, woman is often some more.Fat
Fat tissue is in vivo maximum " energy depot ", the function of fat metabolism gently to be participated in depot lipid, keeping body.Adipose tissue
Energetic supersession is also participated in, and with generation heat, maintenance body temperature, buffer protection and supports the effect such as filling.BAT
In brown, it is characterized in there is abundant capillary in tissue, many small fat drips is dispersed in fat cell, mitochondria is big and rich
Richness, core is circular, and positioned at cell center, this fat cell is also referred to as multilocular adipose cell.
Autologous fat is a kind of preferable packing material, may be used with nearly the soft tissue defects filling at each position of whole body.
However, the maximum defect of fat transfer is exactly the necrosis absorption of adipose tissue, survival rate is not high.Although in order to improve survival rate and
Absorptivity is reduced, adipose tissue volume finally can only after researcher both domestic and external has carried out substantial amounts of research, but current transplanting
It is maintained at during transplanting 50% or so.Therefore, clinically still need by it is a small amount of, repeatedly, repeatedly transplanting improve fat transfer into
Motility rate, to reach the filling effect of patient satisfaction.But multiple fat injection is also made in addition to the workload that increased doctor to patient
Into the pain of multiple liposuction, risk and high expense higher is also undertaken.If can realize once extracting enough fat
External extended refrigerated storage, it is necessary to when rewarming culture after injection transplantation, then with important clinical value.
The content of the invention
In view of this, in order to solve the above problems, freezing for adipose tissue can for a long time be frozen the invention discloses a kind of
Liquid.
The invention discloses a kind of adipose tissue frozen stock solution, based on volumetric concentration, including following component：
Dimethyl sulfoxide (DMSO) 10~20%；
Basal medium 65~90% containing nonessential amino acid；
Dextran 1~20%；
The frozen stock solution is also including trehalose that content is 0.5~1.5g/ml；
Wherein, the ratio of the basal medium and nonessential amino acid is 100:1；
The volumetric concentration summation of all components is 100%.
Preferably, the optimal proportion of the adipose tissue frozen stock solution, based on volumetric concentration, including following component：
Dimethyl sulfoxide (DMSO) 20%；
Basal medium 70% containing nonessential amino acid；
Dextran 5%；
The frozen stock solution also includes that content is the trehalose of 1g/ml；
Wherein, the ratio of the basal medium and nonessential amino acid is 100:1；
The volumetric concentration summation of all components is 100%.
Preferably, the basal medium is DMEM, DMEM/12 or 1640 culture mediums.
Preferably, the dextran is medium molecular dextran, D-40 and Dextran 10
In any one.Wherein, most preferably medium molecular dextran.
Preferably, the trehalose is α, α-type trehalose, α, it is any in β-type trehalose and β, β-type trehalose
It is a kind of.Wherein, most preferably α, α-type trehalose.
The invention also discloses a kind of cryopreservation methods of adipose tissue, comprise the following steps：
Pretreated adipose tissue is put into be made in the cryopreservation tube containing above-mentioned frozen stock solution and freezes sample, then by the jelly
Storage sample product are put into program temperature reduction box, then described program cooling box is put into -80 DEG C of refrigerators, and sample is frozen by described after 24h
Liquid nitrogen is transferred to, completes to freeze.
The invention also discloses application of the adipose tissue frozen stock solution in biological tissue's preservation.
The constituent part that frozen stock solution disclosed by the invention includes is described below：
Dimethyl sulfoxide (DMSO) (DMSO), is a kind of organic compounds containing sulfur, and molecular formula is (CH3)2It is colourless nothing under SO, normal temperature
Smelly transparency liquid, be it is a kind of there is hygroscopic flammable liquid, with highly polar, higher boiling, heat endurance it is good, non-proton,
Characteristic miscible with water, can be dissolved in most of organic matters such as ethanol, propyl alcohol, benzene and chloroform, be referred to as " alembroth ".Deposited in acid
When to DMSO heating can produce the compounds such as a small amount of methyl mercaptan, formaldehyde, dimethyl disulfide and methanesulfonic acid.DMSO has at high temperature
Decomposing phenomenon, meets Hydrogen Energy and vigorous reaction occurs, and burning in atmosphere sends light blue flame.DMSO can be used as organic solvent, anti-
Answer medium and organic synthesis intermediate, it is also possible to make the dyeing solvent of synthetic fibers, remove stain and dyeing carrier, also act as back
Receive the absorbent of acetylene and sulfur dioxide.DMSO can enter cell interior quickly through cell membrane, during cell freezing, dimension
Hold the water and ionic equilibrium of intraor extracellular.
Nonessential amino acid (MEM NEAA), including alanine, arginine, asparatate, cystine, proline, junket
Propylhomoserin etc..Nonessential amino acid can be synthesized by human body itself or amino acid converting be obtained from other.Some nonessential amino acid
Intake can also influence the demand of essential amino acid, for example, when cysteine and abundant tyrosine in meals, can divide
The demand to methionine and phenylalanine is not reduced.Therefore, cysteine and tyrosine are otherwise known as semi-dispensable amino acid or bar
Part essential amino acid.Nonessential amino acid can synthesize in animal body, used as nutrient source, it is not necessary to from external complement amino acid.
Nonessential amino acid is glycine, alanine, proline, tyrosine, serine, cysteine, asparagine, paddy for people
Glutamine, aspartic acid, glutamic acid.These amino acid synthesize carbochain by the metabolin of carbohydrate or by essential amino acid,
Amino generation amino acid is further introduced by transamination reaction.Even if known intake nonessential amino acid, is also have to growth
Profit.
Dextran system sucrose ferments through leuconostoc mesenteroide -1226 (Leuconostoc mesenteroides) and closes
Into a kind of macromolecule glucose polymer, be one of optimal blood substitutes at present.That clinically commonly uses has middle molecule right
The sugared acid anhydride of rotation, is mainly used as blood substitutes, for treating hemorrhagic shock, traumatic shock and burn shock etc..Low, small point
Sub- dextran can improve microcirculation, and prevention or elimination Ink vessel transfusing erythrocyte aggregation and thrombosis etc. also have expanding blood volume
Effect, for treating microcirculation disorder, disseminated intravascular coagulation, angina pectoris, acute myocardial infarction caused by various shocks
And other peripheral vascular diseases etc..
Trehalose also known as cocoon honey, lentinan, are widely distributed a kind of oligosaccharide in animal, plant, microorganism.Work as life
When thing is in adverse circumstances, its content increases, and is that one kind typically stress metabolite.Because it has degeneration-resistant, cold-resistant, drought resisting, resistance to
High temperature, water suction, moisturizing and the features such as protect intracellular large biological molecule so that it food, health products, medicine, cosmetics,
The aspects such as agricultural have very big application prospect.
In the present invention, dextran can be that freezing for tissue block simulates one close to original environment；When fatty group
Knit and lack nutrition supply after leaving body, the supplement to amino acid needs completely, including essential amino acid and nonessential amino acid
Supplement, so just can guarantee that the activity of adipose tissue block；DMSO can enter cell interior quickly through cell membrane, cold in cell
During jelly, maintain the water and ionic equilibrium of intraor extracellular, at the same can with when protection liquid in allow between each component be sufficiently mixed with
And the effect with Synergistic.The characteristics of trehalose has biological protection can further protect the activity of adipose tissue.
The adipose tissue frozen stock solution prepared with the embodiment of the present invention freezes to adipose tissue, will freeze after 3 months
Adipose tissue recover and the form of isolated fat stem cell with freeze before carry out primary isolated fat stem cell
Form is consistent, and compared to traditional frozen stock solution, it is dry thin with the fat after the frozen stock solution cryopreservation resuscitation of embodiment of the present invention preparation
The survival rate of born of the same parents is high, and the immunophenotype and differentiation capability of cell do not change.
In sum, the invention discloses a kind of adipose tissue frozen stock solution, this freezes liquid energy for adipose tissue simulates one
Close to original environment and provide sufficient nutriment, to adipose tissue to freeze effect good, can be long to adipose tissue
Time freezes.The cellular morphology of the isolated fat stem cell of adipose tissue from after cryopreservation resuscitation, immunophenotype and point
Change ability does not change compared with before freezing.And the low cost of the adipose tissue frozen stock solution, beneficial to large-scale promotion application.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is the microscope figure of the fat stem cell that Normal primary is separate；
Primary isolated fat is dry thin after the adipose tissue frozen stock solution cryopreservation resuscitation that Fig. 2 is prepared for the embodiment of the present invention
The microscope figure of born of the same parents；
Fig. 3 is to carry out the streaming result figure of isolated fat stem cell after cryopreservation resuscitation；
Fig. 4 is not freeze the streaming result figure for directly carrying out primary isolated fat stem cell；
Fig. 5 is the external microscope colored graph to Gegenbaur's cell induction differentiation of fat stem cell.
Specific embodiment
The invention discloses a kind of adipose tissue frozen stock solution, the frozen stock solution has to adipose tissue and good freezes effect.
Those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it is all similar
Replacement and change it is apparent to those skilled in the art, they are considered as being included in the present invention.The present invention
Method and application be described by preferred embodiment, related personnel can substantially not depart from present invention, essence
Realize and apply skill of the present invention to method described herein and using being modified or suitably changing with combining in god and scope
Art.
Used in the embodiment of the present invention to preparation be it is commercially available or self-control preparation.
Nonessential amino acid is 100 × solution purchased from GIBCO companies.
Dextran and trehalose are purchased from Sigma companies.
With reference to embodiment, the present invention is expanded on further.
Embodiment 1 prepares adipose tissue frozen stock solution
Wherein, the ratio of DMEM/F12 and nonessential amino acid is 100:1.
Aseptically, the component of above-mentioned amount is mixed, be obtained 2 × frozen stock solution.When actually used by 2 × freeze
Liquid is diluted to 1 × uses.
Embodiment 2 prepares adipose tissue frozen stock solution
Wherein, the ratio of DMEM and nonessential amino acid is 100:1.
Aseptically, the component of above-mentioned amount is mixed, be obtained 2 × frozen stock solution.When actually used by 2 × freeze
Liquid is diluted to 1 × uses.
Embodiment 3 prepares adipose tissue frozen stock solution
Wherein, the ratio of 1640 culture mediums and nonessential amino acid is 100:1.
Aseptically, the component of above-mentioned amount is mixed, be obtained 2 × frozen stock solution.When actually used by 2 × freeze
Liquid is diluted to 1 × uses.
The adipose tissue of embodiment 4 freezes experiment
The frozen stock solution prepared using embodiment 1 as test group, using traditional frozen stock solution as a control group.Tradition freezes
The formula of liquid adds the DMSO of 20 units for the FBS of 80 units.
20 parts of adipose tissue of collection (is authorized by volunteer and agreed to, 20~30 years old age, non-communicable disease), will be adopted
The adipose tissue for collecting is equally divided into two parts, and a part carries out primary separation, and the cell culture for obtaining to P2 generations is frozen；
A part is cleaned screening and obtains adipose tissue with following methods, and the adipose tissue that will be obtained is equally divided into two parts, and a part is used
The adipose tissue frozen stock solution of test group freezes, and the frozen stock solution of another part control group freezes.
To the cleaning screening technique (pretreatment) of adipose tissue：After cleaning 2 times using isometric physiological saline 500g from
Heart 5min, abandons upper strata oil layer and lower floor's physiology salt water layer, and 2mm screen clothes are crossed after being shredded with scissors, and the adipose tissue for obtaining is for most
The adipose tissue for freezing eventually.
Above-mentioned pretreated adipose tissue is put into be made in the cryopreservation tube containing 1ml frozen stock solutions and freezes sample, will prepared
The good sample that freezes is put into program temperature reduction box, then program temperature reduction box is put into -80 DEG C of refrigerators, and finished product is transferred into liquid after 24h
In nitrogen, complete to freeze, can't directly place products into liquid nitrogen.
The fat stem cell that Normal primary is separate is observed under inverted microscope, as a result as shown in figure 1, Figure 1A is
The P2 fat subsitutes stem cell that Normal primary is separate amplifies 40 times of microscope figure, and Figure 1B is that the P2 fat subsitutes that Normal primary is separate are done
Cell amplifies 100 times of microscope figure.Normal human adipose-derived stem cell adherent growth, is the relatively uniform spindle cell of form.
Above-mentioned Normal primary separation method is type i collagen enzyme digestion：By pretreated adipose tissue with final concentration of
After 0.2% type i collagen enzymic digestion 1h, 1500rpm centrifugation 5min, centrifugation uses the resuspended inoculation of culture medium after terminating.
The adipose tissue block for freezing is taken out after freezing 3 months, it is dry to fat using the primary separation method with before
Cell is separated, and statistics is as follows：
Group obtains P0 cell success rates
Test group 20 100%
Control group 12 60%
Observed under inverted microscope for cell carrying out being separately cultured the P2 for obtaining after recovery, as a result such as Fig. 2
Shown, Fig. 2A amplifies 40 times of microscope figure for P2 fat subsitutes stem cell isolated after test group cryopreservation resuscitation, and Fig. 2 B are
P2 fat subsitutes stem cell isolated after test group cryopreservation resuscitation amplifies 100 times of microscope figure.By comparing Fig. 1 and Fig. 2
As can be seen that fat stem cell isolated after test group cryopreservation resuscitation with without freezing direct primary isolated fat
Stem cell morphology is consistent, and the success rate of isolated fat stem cell is higher than control group after test group cryopreservation resuscitation, it is seen that
The frozen stock solution of test group is better than control group to the effect that freezes of adipose tissue.
The frozen stock solution prepared with embodiment 2 or 3 repeats above-mentioned experiment, obtains similar result.
The Immunophenotype analysis of isolated cell are carried out after the cryopreservation resuscitation of embodiment 5
Test group：After the test group of embodiment 4 (frozen stock solution that embodiment 1 is prepared) is frozen into recovery in 3 months separate
The fat stem cell culture arrived is cleaned 3 times to the 3rd generation after being digested to cell, is made cell suspension, and stream is used after adding antibody
Formula cell instrument tests and analyzes the expression of CD73, CD90, CD105, CD11b, CD19, CD34, CD45, HLA-DR.
Control group：Primary isolated fat stem cell is directly carried out without the adipose tissue block for freezing, is entered with test group
The same flow cytometer showed of row.
The flow cytometer showed result of test group and control group is compared, the streaming result of test group is shown in Fig. 3 and table 1：
The test group streaming result of table 1
The streaming result of control group is shown in Fig. 4 and table 2：
The control group streaming result of table 2
Flow cyctometry detection display, test group does not express the moon as the cell marker expression of control group
Cell marker thing (hematopoietic cell markers CD34, CD45, CD11, HLA-DR and CD19)；And positive cell label is in height
Expression (integrin and adhesion molecule CD90, and conventional human mesenchymal stem cell mark CD73 and CD105), and test group and
Control group is as good as (P to the expression of negative cells label and positive cell label>0.05).Illustrate that embodiment 1 is prepared
Frozen stock solution freeze after adipose tissue maintain its original activity well, fat stem cell and the fresh fat group of acquisition
Knit and be as good as.
Frozen stock solution prepared by Example 2 or 3 repeats above-mentioned experiment, obtains similar result.
Isolated cells in vitro is carried out after the cryopreservation resuscitation of embodiment 6 to osteocyte induction differentiation detection
Induction experiment group：Divided after the test group of embodiment 4 (frozen stock solution that embodiment 1 is prepared) is frozen into recovery in 6 months
From the fat stem cell culture for obtaining to logarithmic phase P5 generations are grown, it is inoculated in six orifice plates, is respectively provided with control group and induction group,
1.0*10 is per hole cell-seeding-density4/ hole, adds 2mlDMEM/F12 complete mediums to be cultivated per hole.Treat cell fusion
To 60-70%, old culture medium is discarded, change bone induction culture medium into, per hole liquid feeding 3ml, change liquid once within every 3 days；Induction training
Support during by the 4th week, carry out skeletonization dyeing to the cell of induction group and control group with Alizarin red staining liquid and identify and gather image.
Induction control group：Primary isolated fat stem cell is directly carried out without the tissue block for freezing, with induction experiment
Group carries out the same external detection to Gegenbaur's cell induction differentiation.
Control group：After the test group of embodiment 4 (frozen stock solution that embodiment 1 is prepared) is frozen into recovery in 6 months separate
To fat stem cell carry out normal culture to growth logarithmic phase P5 generations, cultural method isogeneous induction test group.But in cell fusion
During to 60~70%, after discarding old culture medium, the low sugar DMEM culture mediums containing 5%FBS are changed into, follow-up cultivation and colouring method are same
Induction experiment group, the external induction to Gegenbaur's cell is not carried out and is broken up.
Result is as shown in Figure 5.
Testing result shows that the fat stem cell of induction experiment group and induction control group can be thin through induced synthesis skeletonization
Born of the same parents, have substantial amounts of doped calcium in the cytoplasm of Alizarin red staining discovery induction experiment group, illustrate that the tissue prepared by embodiment 1 freezes
The cell that obtains of adipose tissue block after liquid storage freezes has normal differentiation capability.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (7)
1. a kind of adipose tissue frozen stock solution, it is characterised in that based on volumetric concentration, including following component：
Dimethyl sulfoxide (DMSO) 10~20%；
Basal medium 65~90% containing nonessential amino acid；
Dextran 1~20%；
The frozen stock solution is also including trehalose that content is 0.5~1.5g/ml；
Wherein, the ratio of the basal medium and the nonessential amino acid is 100:1；
The volumetric concentration summation of all components is 100%.
2. adipose tissue frozen stock solution according to claim 1, it is characterised in that based on volumetric concentration, including following component：
The frozen stock solution also includes that content is the trehalose of 1g/ml；
Wherein, the ratio of the basal medium and the nonessential amino acid is 100:1；
The volumetric concentration summation of all components is 100%.
3. frozen stock solution according to claim 1 and 2, it is characterised in that the basal medium be DMEM, DMEM/12 or
1640 culture mediums.
4. frozen stock solution according to claim 1 and 2, it is characterised in that the dextran is medium molecular dextran, low
Any one in molecule dextran and Dextran 10.
5. frozen stock solution according to claim 1 or claim 2, it is characterised in that the trehalose is α, α-type trehalose, α, β-type sea
Algae sugar and β, β-type trehalose in any one.
6. a kind of adipose tissue cryopreservation methods, it is characterised in that comprise the following steps：
Pretreated adipose tissue is put into the cryopreservation tube containing the frozen stock solution described in claim 1 to 5 any one and is made
Sample is frozen, then the sample that freezes is put into program temperature reduction box, then described program cooling box is put into -80 DEG C of refrigerators
It is interior, the sample that freezes is transferred to liquid nitrogen after 24h, complete to freeze.
7. application of the adipose tissue frozen stock solution described in claims 1 to 3 any one in biological tissue's preservation.
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| CN108795853A (en) * | 2018-05-28 | 2018-11-13 | 天津博雅秀岩生物技术有限公司 | Prepare the method and dog fetal membrane mescenchymal stem cell of dog fetal membrane mescenchymal stem cell |
| CN109221089A (en) * | 2018-10-16 | 2019-01-18 | 浙江美泰生物科技有限公司 | A kind of method that the frozen stock solution and adipose tissue of adipose tissue freeze |
| CN109497041A (en) * | 2018-11-23 | 2019-03-22 | 西安九州医学中心有限公司 | A kind of cryopreservation methods of adipose tissue |
| CN109526941A (en) * | 2018-12-27 | 2019-03-29 | 广州赛莱拉干细胞科技股份有限公司 | A kind of preservation liquid of fat mesenchymal stem cell |
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