CN108719275A - A kind of preservation liquid and its application method for storage in vitro lipochondrion - Google Patents
A kind of preservation liquid and its application method for storage in vitro lipochondrion Download PDFInfo
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- CN108719275A CN108719275A CN201810576523.9A CN201810576523A CN108719275A CN 108719275 A CN108719275 A CN 108719275A CN 201810576523 A CN201810576523 A CN 201810576523A CN 108719275 A CN108719275 A CN 108719275A
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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
A kind of preservation liquid and its application method for storage in vitro lipochondrion, the present invention relates to a kind of preservation liquid and its application method for storage in vitro lipochondrion, the purpose of the present invention is to solve the low problems of the free SVF cell activity in the fat blend preserved in existing preservation liquid, and the present invention, which preserves liquid, to be mixed by penicillin, streptomysin, low molecular sodium heparin, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride.Application method is：It is 1 by volume by lipochondrion:3 ratio is added to preserve and be preserved in liquid.It is preserved in liquid in the present invention and preserves 48h, be maintained to the high activity of SVF in adipocyte, expanding capacity and differentiation function.The present invention is applied to lipochondrion preservation field.
Description
Technical field
The present invention relates to a kind of preservation liquid and its application method for storage in vitro lipochondrion.
Background technology
Adipose tissue is easy a large amount of acquisitions with it, does not influence for area's function, excellent without immunological rejection etc. after autotransplantation
Point becomes the theoretic preferred filler of soft tissue defects.Clinical application has longer history, and the research of nineteen fifty Peer is found
The lipochondrion of transplanting reduces 50% or more mostly on volume and weight, and downright bad lipochondrion often causes fibrocystic
Change and false cyst.In addition, there is also the complication such as liquefaction, necrosis, infection in the treatment, adipose tissue is hindered as soft group
Knit the application of filler clinically.With the development of organizational project, is received and ground at adipose tissue using stem cell Immune inducing in vivo
The concern for the person of studying carefully, if technique can be successfully applied to the clinical tradition that will solve and transplant the various of mature fat cell.Self
Fat transfer is a kind of material that Soft-tissue operation is best, may be used with nearly the soft tissue defects filling at each position of whole body, to the greatest extent
For pipe in order to improve survival rate and reduce absorptivity, domestic and international researcher has carried out a large amount of research, but fat after transplanting at present
Tissue body tissue can only keep 50% or so when transplanting.Therefore, clinically need by it is a small amount of, repeatedly, repeatedly transplanting improve
Fat survival ensures the activity of adipocyte, to reach satisfied filling effect.
Also contain a large amount of mescenchymal stem cell (adipose tissue- with multi-lineage potential in adipose tissue
Derived mesenchymal stem cells, ASCs), the adipose tissue remained on after adipose tissue extraction for area can still expand
Increase, the core technology of the fat transfer technology (cell-assisted lipotransfer, CAL) of cell auxiliary is to utilize purification
The stromal vascular layer (stromal vascular fraction, SVF) containing adipose-derived stem cells (ADSC) and particle fat
Fat tissue mixed transplantation carries out Soft-tissue operation, and stem cell can be divided into the adipocyte of a part after transplanting, with traditional
Transplant fat cell is compared, and has better survival rate and quantity to maintain effect also more preferable.SVF is obtained from liposuction technique
Fat blend, the SVF in adipose tissue is made of various kinds of cell group, further includes Hematopoietic Stem in addition to fat stem cell
Cell, fibrocyte, endothelial cell, Preadipocyte In Vitro and macrophage etc..After SVF can be by collagenase digesting adipose tissue
Centrifugation acquisition, can be used for autologous fat transplantation, reduces the absorptivity of transplanting tissue, maintains volume and reduction complication after transplanting.
Can also by adhere-wall culture can therefrom separating-purifying ADSC be used for regenerative medicine research.
After routine liposuction at present, fat blend is transferred to containing in the preservation bottle for preserving liquid (mainly physiological saline)
Of short duration preservation is preserved at this and is preserved in liquid, and the free SVF activity in fat blend is low, illustrates that the preservation liquid energy is enough destroyed
Cell, to also reduce fat stem cell content.
Invention content
The purpose of the present invention is to solve the free SVF cells work in the fat blend preserved in existing preservation liquid
The low problem of property, provides a kind of preservation liquid and its application method for storage in vitro lipochondrion.
A kind of preservation liquid for storage in vitro lipochondrion of the invention be by penicillin, streptomysin, low molecular sodium heparin,
Sodium gluconate, sodium acetate, potassium chloride and magnesium chloride mix, pH 7.0-7.5；It wherein preserves per 1000mL and contains in liquid
There are NaCl, 5.0- of the penicillin of 5-15mL, the streptomysin of 5-15mL, the low molecular sodium heparin of 150-200 μ L, 5.3-5.7g
The magnesium chloride of the sodium gluconate of 5.5g, the potassium chloride of 3.6-4.0g, 0.2-0.4g.
A kind of application method of preservation liquid for storage in vitro lipochondrion of the invention is：By volume by lipochondrion
It is 1:3 ratio is added to preserve and be preserved in liquid.
Advantageous effect of the present invention：
Preserved in liquid in the present invention and preserve 48h, be maintained to the high activity of SVF in adipocyte, expanding capacity and
Differentiation function；
(1) show its SVF activity of the lipochondrion preserved in this preservation liquid higher than other preservation liquid by experimental study
Fatty granule cell (the P of preservation<0.01) SVF activity is up to 93.73% ± 0.14 after, preserving 48h；
(2) show that the doubling time of its adipocyte of preservation lipochondrion in this preservation liquid is less than by experimental study
Other preserve the fatty granule cell that liquid preserves, the relatively strong (P of expanding capacity<0.01), the doubling time is 40.1h ± 1.23；
(3) lipochondrion that liposuction source can be largely preserved with this preservation liquid, fills out and fills for the benefit in the short time
Other positions.
Description of the drawings
Fig. 1 is that different preservation liquid preserve the adherent situation of fat stem cell after lipochondrion；
Fig. 2 is the testing result that different preservation liquid preserve cell differentiation functional after lipochondrion.
Specific implementation mode
Technical solution of the present invention is not limited to the specific implementation mode of act set forth below, further include each specific implementation mode it
Between arbitrary combination.
Specific implementation mode one：A kind of preservation liquid for storage in vitro lipochondrion of present embodiment be by penicillin,
Streptomysin, low molecular sodium heparin, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride mix, pH 7.0-7.5；Its
In per 1000mL preserve liquid in the penicillin containing 5-15mL, the streptomysin of 5-15mL, 150-200 μ L low molecular sodium heparin,
The magnesium chloride of the sodium gluconate of NaCl, 5.0-5.5g of 5.3-5.7g, the potassium chloride of 3.6-4.0g, 0.2-0.4g.
Present embodiment advantageous effect：
Preserved in liquid in the present invention and preserve 48h, be maintained to the high activity of SVF in adipocyte, expanding capacity and
Differentiation function can carry out mending again filling out or filling other positions, avoid liposuction again, reduce the pain of patient with remaining fat
It is bitter；
(1) show its SVF activity of the lipochondrion preserved in this preservation liquid higher than other preservation liquid by experimental study
Fatty granule cell (the P of preservation<0.01) SVF activity is up to 93.73% ± 0.14 after, preserving 48h；
(2) show that the doubling time of its adipocyte of preservation lipochondrion in this preservation liquid is less than by experimental study
Other preserve the fatty granule cell that liquid preserves, and the doubling time is 40.1h ± 1.23, the relatively strong (P of expanding capacity<0.01)；
(3) lipochondrion that liposuction source can be largely preserved with this preservation liquid, fills out and fills for the benefit in the short time
Other positions.
Specific implementation mode two：The present embodiment is different from the first embodiment in that：It preserves per 1000mL and contains in liquid
There is the penicillin of 10mL.Other are the same as one or two specific embodiments.
Specific implementation mode three：The present embodiment is different from the first and the second embodiment in that：Liquid is preserved per 1000mL
In the streptomysin containing 10mL.It is the same as one or two specific embodiments.
Specific implementation mode four：Unlike one of present embodiment and specific implementation mode one to three：It is protected per 1000mL
Low molecular sodium heparin containing 180 μ L in liquid storage.Other are identical as one of specific implementation mode one to three.
Specific implementation mode five：Unlike one of present embodiment and specific implementation mode one to four：It is protected per 1000mL
NaCl containing 5.6g in liquid storage.Other are identical as one of specific implementation mode one to four.
Specific implementation mode six：Unlike one of present embodiment and specific implementation mode one to five：It is protected per 1000mL
Sodium gluconate containing 5.3g in liquid storage.Other are identical as one of specific implementation mode one to five.
Specific implementation mode seven：Unlike one of present embodiment and specific implementation mode one to six：It is protected per 1000mL
Potassium chloride containing 3.8g in liquid storage.Other are identical as one of specific implementation mode one to six.
Specific implementation mode eight：Unlike one of present embodiment and specific implementation mode one to seven：It is protected per 1000mL
Magnesium chloride containing 0.3g in liquid storage.Other are identical as one of specific implementation mode one to seven.
Specific implementation mode nine：Present embodiment is unlike specific implementation mode seven or eight：It is adjusted and is protected using NaOH
The pH of liquid storage.Other are identical as specific implementation mode seven or eight.
Specific implementation mode ten：A kind of application method of preservation liquid for storage in vitro lipochondrion of present embodiment
For：It is 1 by volume by lipochondrion:3 ratio is added to preserve and be preserved in liquid.
Beneficial effects of the present invention are verified using following embodiment：
Embodiment one：A kind of preservation liquid for storage in vitro lipochondrion is by penicillin, streptomysin, low molecular weight heparin
Sodium, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride mix, pH 7.0-7.5；Liquid is wherein preserved per 1000mL
In the penicillin containing 5-15mL, the streptomysin of 5-15mL, the low molecular sodium heparin of 150-200 μ L, 5.3-5.7g NaCl,
The magnesium chloride of the sodium gluconate of 5.0-5.5g, the potassium chloride of 3.6-4.0g, 0.2-0.4g.
Containing 1% dual anti-physiological saline (preserving liquid 1), it is dual anti-to contain 1% respectively for the lipochondrion that liposuction is obtained
Serum free medium (preserve liquid 2) and the present invention preservation liquid in preserved, the volume ratio of lipochondrion and preservation liquid is
1:3；Liquid and both the above difference are preserved to the present invention and preserve the fat tissue cell and SVF cell activity that liquid preserves, is proliferated work(
Can, surface markers and differentiation function etc. are detected.By FCM analysis express typical MSCs surface antigens CD73,
CD90, CD105, positive expression rate reach 99% or more, do not express hemopoietic stem cell surface antigen CD4 5, CD14, CD34,
CD79a and surface endothelial cell antigens, feminine gender expression are less than 2% (being shown in Table 1).
Table 1：Difference preserves the influence to fat stem cell surface markers after liquid preservation lipochondrion expands
Table 2 is the influence detection that different preservation liquid preserve SVF cell activity after lipochondrion, as can be drawn from Table 2, at this
Invention, which preserves, preserves 48h in liquid, be maintained to the high activity of SVF in adipocyte, and SVF activity can reach 93.73% ±
0.14。
Table 2：Difference preserves the influence detection that liquid preserves SVF cell activity after lipochondrion
| Difference preserves liquid | Preserve 12h | It preserves for 24 hours | Preserve 48h |
| Preserve liquid 1 | 85.41% ± 0.21 | 82.50% ± 0.20 | 77.45% ± 0.30 |
| Preserve liquid 2 | 84.53% ± 0.09 | 80.71% ± 0.21 | 75.62% ± 0.21 |
| This preservation liquid | 98.28% ± 0.12 | 96.21% ± 0.19 | 93.73% ± 0.14 |
Show that the doubling time of its adipocyte of preservation lipochondrion in this preservation liquid is less than other by experimental study
The fatty granule cell (table 3) that liquid preserves is preserved, the doubling time is 40.1h ± 1.23, illustrates to preserve in the present invention preserves liquid
The relatively strong (P of lipocyte proliferation function<0.01).
The different liquid that preserve of table 3 preserve fat stem cell expanding capacity detection after lipochondrion
Difference preserves after liquid preserve lipochondrions that the adherent situation of fat stem cell is as shown in Figure 1, as shown in Figure 1, the present invention
Fat stem cell is adherent after preservation liquid preserves will be obviously for preserving liquid 1 and preserving liquid 2.After difference preserves liquid preservation lipochondrion
As shown in Fig. 2, as shown in Figure 2, it is apparent at differentiation is referred to that the present invention preserves cell after liquid preserves for the testing result of cell differentiation functional,
Illustrate that the present invention preserves liquid and is less than preservation liquid 1 to the influence of the differentiation function of cell and preserves liquid 2.
Claims (10)
1. a kind of preservation liquid for storage in vitro lipochondrion, it is characterised in that the preservation liquid is by penicillin, streptomysin, low
Molecular sodium heparin, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride mix, pH 7.0-7.5；It is wherein every
1000mL preserves low molecular sodium heparin, the 5.3- of the penicillin containing 5-15mL, the streptomysin of 5-15mL, 150-200 μ L in liquid
The magnesium chloride of the sodium gluconate of NaCl, 5.0-5.5g of 5.7g, the potassium chloride of 3.6-4.0g, 0.2-0.4g.
2. a kind of preservation liquid for storage in vitro lipochondrion according to claim 1, it is characterised in that per 1000mL
Preserve the penicillin containing 10mL in liquid.
3. a kind of preservation liquid for storage in vitro lipochondrion according to claim 1, it is characterised in that per 1000mL
Preserve the streptomysin containing 10mL in liquid.
4. a kind of preservation liquid for storage in vitro lipochondrion according to claim 1, it is characterised in that protected per 1000mL
Low molecular sodium heparin containing 180 μ L in liquid storage.
5. a kind of preservation liquid for storage in vitro lipochondrion according to claim 1, it is characterised in that per 1000mL
Preserve the NaCl containing 5.6g in liquid.
6. a kind of preservation liquid for storage in vitro lipochondrion according to claim 1, it is characterised in that per 1000mL
Preserve the sodium gluconate containing 5.3g in liquid.
7. a kind of preservation liquid for storage in vitro lipochondrion according to claim 1, it is characterised in that per 1000mL
Preserve the potassium chloride containing 3.8g in liquid.
8. a kind of preservation liquid for storage in vitro lipochondrion according to claim 1, it is characterised in that per 1000mL
Preserve the magnesium chloride containing 0.3g in liquid.
9. a kind of preservation liquid for storage in vitro lipochondrion according to claim 1, it is characterised in that use NaOH
Adjust the pH for preserving liquid.
10. a kind of application method of preservation liquid for storage in vitro lipochondrion as described in claim 1, it is characterised in that
The application method of preservation liquid for storage in vitro lipochondrion is：It is 1 by volume by lipochondrion:3 ratio is added to
It preserves and is preserved in liquid.
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| CN201810576523.9A CN108719275A (en) | 2018-06-06 | 2018-06-06 | A kind of preservation liquid and its application method for storage in vitro lipochondrion |
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| CN109430251A (en) * | 2018-12-18 | 2019-03-08 | 广州康琪莱生物科技有限公司 | A kind of store method of the preservation liquid of adipose tissue and preparation method thereof with adipose tissue |
| CN109662091A (en) * | 2019-03-01 | 2019-04-23 | 米楠 | A kind of lipochondrion tissue freezing solution and its preparation method and cryopreservation methods |
| CN109769799A (en) * | 2019-03-20 | 2019-05-21 | 江苏瑞思坦生物科技有限公司 | A kind of human fat tissue saves liquid and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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Application publication date: 20181102 |