CN109497038A - A kind of fat saves liquid and preparation method thereof - Google Patents
A kind of fat saves liquid and preparation method thereof Download PDFInfo
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- CN109497038A CN109497038A CN201710828614.2A CN201710828614A CN109497038A CN 109497038 A CN109497038 A CN 109497038A CN 201710828614 A CN201710828614 A CN 201710828614A CN 109497038 A CN109497038 A CN 109497038A
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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 invention discloses a kind of fat to save liquid, for saving adipose tissue sample, main component is DMEM in high glucose dehydrated medium, sodium bicarbonate, insulin, penicillin, streptomysin, anphotericin, aspirin, vitamin C, resveratrol, icariin, hydrocortisone, VEGF, IGF-I, EPO.Fat of the invention saves liquid and is easy preparation, low in cost, easy to use, can keep the activity of stem cell in adipose tissue for a long time, greatly reduce tissue samples from the time restriction for collecting preparation, and contamination probability can be effectively reduced.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of preservation liquid for saving fatty sample.
Background technique
Mescenchymal stem cell (MSCs) is a kind of stem cell with multi-lineage potential, has and is divided into a variety of mesoderms
The potential of cell line, including osteoblast, fat cell, cartilage cell etc..During body normal injury repair, MSCs
It can recruit under the induction of chemotactic factor (CF) to damage location, participate in damaging in local multiplication, differentiation, and by paracrine action
Wound is repaired and regeneration.The MSCs of adipose tissue-derived is easily isolated and amplification in vitro, by repeatedly divide passage after still
Multi-lineage potential is kept, is the seed cell of a kind of ideal cell therapy and regenerative medicine.Adipose tissue distribution is wide
General, acquisition is easy, and no ethics problem acquires basic no pain, and can be with multi collect.
Fatty MSCs preparation process includes the acquisition of tissue samples, transport, handover, detection, separates, freezes, recovering, is primary
All multi-steps such as culture and secondary culture need to transport to before separating after fatty sample collection, three steps of handover and detection,
It needs to store longer time in practical operation.Once adipose tissue sample is therein departing from original vivo environment acquisition
The activity of MSCs can be influenced by many factors, time, temperature, osmotic pressure etc..The many situations of adipose tissue sample at present
Under need strange land to acquire, transport, join and detect the time used in three steps with regard to longer, the time that tissue samples need to be stored compared with
Long, influence factor is just more complicated.Domestic field of biology to the preservation of adipose tissue sample mostly use directly bottling or
The method for adding basal medium.The holding time of directly bottling adipose tissue sample is generally less than 24 hours, gives adipose tissue
Transport, handover and the detection of sample bring no small time pressure, and there is an urgent need to improve its store method.
Summary of the invention
The purpose of the invention is to overcome in the prior art, the fatty sample holding time is short, stem cell pick-up rate and activity
Impacted problem provides a kind of fat preservation liquid, which saves liquid proportion simply, low in cost, easy to use.It utilizes
Fat of the invention, which saves liquid, can save the activity of stem cell in adipose tissue sample, effectively saved in the case where 0 ~ 8 DEG C to
Few 120 hours, the Stem Cell Activity isolated was influenced very little by the time, greatly reduced tissue samples from collecting preparation
Time restriction, and contamination probability can be effectively reduced.
In order to achieve the above object, the technical solution adopted by the present invention is that: it is a kind of fat save liquid, every liter fat save liquid
It is made of by weight following ingredient: DMEM in high glucose dehydrated medium 13.4g, sodium bicarbonate 2.438g, 2~10mg of insulin, blueness
100~200mg of mycin, 100~200mg of streptomysin, 2.5~5mg of anphotericin, 200 ~ 400mg of aspirin, vitamin C
300~700mg, 7~12mg of resveratrol, 2~10mg of icariin, 20~40ug of hydrocortisone, 10~20 μ g of VEGF,
5~10 μ g of IGF-I, 5 EPO~10 μ g, surplus is water for injection.
A kind of above-mentioned fat saves liquid, and preparation method includes the following steps:
(1) DMEM in high glucose dehydrated medium 13.4g is poured into a container, with a small amount of water for injection by remaining medium in bag
It washes down, is incorporated to container.Inject water to 950ml, gentle agitation dissolution；
(2) 2.438 g sodium bicarbonates are added；
(3) gentle agitation dissolves, and injects water to 1L；
(4) pH value is adjusted to neutrality with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution；
(5) 2~10mg of insulin, 100~200mg of penicillin, 100~200mg of streptomysin, anphotericin are successively separately added into
2.5~5mg, 200 ~ 400mg of aspirin, 300~700mg of vitamin C, 7~12mg of resveratrol, icariin 2~
10mg, 20~40ug of hydrocortisone, 10~20 μ g of VEGF, 5 IGF-I~10 μ g, 5 EPO~10 μ g, mix well；
(6) degerming of 0.22um membrane filtration is made present invention fat and saves liquid, packing, freezen protective；It is placed in 0 when use in advance ~
8 DEG C of defrostings.
Fat of the invention saves liquid, wherein DMEM in high glucose and sodium bicarbonate be maintain the osmotic equilibrium of cell inside and outside tissue,
PH value is maintained, adipose tissue moisture state is kept and nutritional ingredient is provided；Penicillin, streptomysin, anphotericin can prevent bacterium,
The pollution such as mould, and the pollution occurred can be eliminated；Vitamin C, resveratrol, icariin are antioxidants, can keep rouge
Fat activity；Hydrocortisone inhibits immune, protects Stem Cell Activity in adipose tissue sample；Insulin promotes suction of the fat to sugar
It receives and utilizes；Cell factor such as VEGF, IGF-I, EPO can keep increasing Stem Cell Activity in adipose tissue.
It is easy that fat of the invention saves liquid preparation, low in cost, easy to use, the tissue samples after making acquisition transport,
Before handover, separation during detection etc., the activity of wherein stem cell can be effectively kept in a long time, and can prevent and go
Depollution improves efficiency.
Specific embodiment
Invention is further described in detail below.
Embodiment one
A kind of fat provided by the invention saves liquid, and every liter of fat saves liquid and is made of by weight following ingredient: DMEM in high glucose is dry
Powder culture medium 13.4g, sodium bicarbonate 2.438g, insulin 10mg, penicillin 200mg, streptomysin 200mg, anphotericin 5mg,
Aspirin 400mg, vitamin C 700mg, resveratrol 12mg, icariin 10mg, hydrocortisone 40ug, VEGF
20 μ g, 10 IGF-I μ g, 10 EPO μ g, surplus is water for injection.
The fat save liquid the preparation method is as follows:
(1) DMEM in high glucose dehydrated medium (brand: GIBCO, article No. 12100-046) 13.4g is poured into a container, with less
Amount water for injection washes down remaining medium in bag, is incorporated to container.Inject water to 950ml, gentle agitation dissolution；
(2) 2.438 g sodium bicarbonates are added；
(3) gentle agitation dissolves, and injects water to 1L；
(4) pH value is adjusted to neutrality with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution；
(5) insulin 10mg(brand Sigma, article No. I0908 are successively separately added into), penicillin 200mg(brand Amresco,
Article No. 0242), streptomysin 200mg(brand Amresco, article No. 0382), anphotericin 5mg(brand Amresco, article No.
E437), aspirin 400mg(Sigma, A2093-100G), vitamin C 700mg(Sigma, A5960-10mg), white black false hellebore
Alcohol 12mg(Sigma, R5010-100MG), the Shanghai icariin 10mg(crystallite biology, 489-32-7,20mg), hydrogenation can
Loose 40ug(Sigma, H3160), VEGF 20 μ g(PeproTech, 96-100-20-2), 10 μ g(PeproTech of IGF-I,
96-100-11-20), 10 μ g(PeproTech, CYT-201 of EPO), it mixes well；
(6) 0.22um membrane filtration degerming is made fat and saves liquid, packing, freezen protective；0 ~ 8 DEG C of solution is placed in when use in advance
Jelly.
Embodiment two
A kind of fat provided by the invention saves liquid, and every liter of fat saves liquid and is made of by weight following ingredient: DMEM in high glucose is dry
Powder culture medium 13.4g, sodium bicarbonate 2.438g, insulin 2mg, penicillin 100mg, streptomysin 100mg, anphotericin 2.5mg,
Aspirin 200mg, vitamin C 300mg, resveratrol 7mg, icariin 2mg, hydrocortisone 20ug, VEGF 10
μ g, 5 IGF-I μ g, 5 EPO μ g, surplus is water for injection.
The fat save liquid the preparation method is as follows:
(1) DMEM in high glucose dehydrated medium 13.4g is poured into a container, with a small amount of water for injection by remaining medium in bag
It washes down, is incorporated to container.Inject water to 950ml, gentle agitation dissolution；
(2) 2.438 g sodium bicarbonates are added；
(3) gentle agitation dissolves, and injects water to 1L；
(4) pH value is adjusted to neutrality with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution；
(5) insulin 2mg, penicillin 100mg, streptomysin 100mg, anphotericin 2.5mg, aspirin are successively separately added into
200mg, vitamin C 300mg, resveratrol 7mg, icariin 2mg, 10 μ g of hydrocortisone 20ug, VEGF, IGF-I
5 μ g, 5 EPO μ g, mix well；
(6) 0.22um membrane filtration degerming, packing, freezen protective are made fat and save liquid；0 ~ 8 DEG C of solution is placed in when use in advance
Jelly.
Embodiment three
A kind of fat provided by the invention saves liquid, and every liter of fat saves liquid and is made of by weight following ingredient: DMEM in high glucose is dry
Powder culture medium 13.4g, sodium bicarbonate 2.438g, insulin 5mg, penicillin 150mg, streptomysin 150mg, anphotericin 4mg, Ah
Take charge of a woods 300mg, vitamin C 400mg, resveratrol 8mg, icariin 3mg, 15 μ of hydrocortisone 30ug, VEGF
G, 6 μ g of IGF-I, 6 EPO μ g, surplus is water for injection.
The fat save liquid the preparation method is as follows:
(1) DMEM in high glucose dehydrated medium 13.4g is poured into a container, with a small amount of water for injection by remaining medium in bag
It washes down, is incorporated to container.Inject water to 950ml, gentle agitation dissolution；
(2) 2.438 g sodium bicarbonates are added；
(3) gentle agitation dissolves, and injects water to 1L；
(4) pH value is adjusted to neutrality with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution；
(5) insulin 5mg, penicillin 150mg, streptomysin 150mg, anphotericin 4mg, aspirin are successively separately added into
300mg, vitamin C 400mg, resveratrol 8mg, icariin 3mg, 15 μ g of hydrocortisone 30ug, VEGF, IGF-I
6 μ g, 6 EPO μ g, mix well；
(6) degerming of 0.22um membrane filtration is made fat and saves liquid, packing, freezen protective；0 ~ 8 DEG C of solution is placed in when use in advance
Jelly.
Example IV
The acquisition and preservation of adipose tissue: being saved after liquid saves liposuction with fat of the invention and arrive the human fat tissue before separating,
It i.e. after aseptic collection fat, inhales and abandons inflation fluid, the adipose tissue of acquisition is put into the fat for saving liquid equipped with fat of the invention
It in collecting bottle, tightens bottle cap and seals, be put into 0 ~ 8 DEG C of constant temperature in insulating box and save to separation.
It saves the preservation effect comparison of liquid: the adipose tissue of acquisition being divided into 5 parts, is stored in fat of the invention respectively
It saves in liquid, phosphate buffer PBS, physiological saline and DMEM in high glucose culture medium (liquid), last portion is directly by fatty group
Knit individually placed, collagenase digesting separates after 0 ~ 8 DEG C of constant temperature saves 24 hours, 48 hours, 72 hours, 120 hours, takes identical
Behind karyocyte routine culture 9 days that the separation of grams adipose tissue block obtains, digestion is counted.A fresh fat tissue is stayed simultaneously
It does not save as control, the karyocyte routine culture for taking identical grams adipose tissue block to obtain after direct collagenase digesting separation
9 days, 6.20 × 10 are counted as after digestion7。
It saves as follows as the result is shown:
Past 1 year fat acquisition is transported and carries out pollution rate statistics, being not added in 64 parts of adipose tissue that any solution saves has 2
Part pollution, pollution rate 3.1%；It saves in 371 parts of adipose tissues that liquid saves through fat of the invention without a pollution, pollution
Rate is 0.
As can be seen that fat of the invention saves liquid, the tissue samples after can making acquisition are before transport, handover, separation
During detection etc., the activity of wherein stem cell can be effectively kept (more than 5 days) in a long time, stem cell is improved and obtains
Rate, and depollution can be prevented and go, expand the scope of business of tissue samples library stem cell storage.
Claims (2)
1. a kind of fat saves liquid, characterized in that every liter of fat saves liquid and is made of by weight following ingredient: DMEM in high glucose dry powder
Culture medium 13.4g, sodium bicarbonate 2.438g, 2~10mg of insulin, 100~200mg of penicillin, 100~200mg of streptomysin, two
2.5~5mg of property mycin, 200 ~ 400mg of aspirin, 300~700mg of vitamin C, 7~12mg of resveratrol, icariin 2
~10mg, 20~40ug of hydrocortisone, 10~20 μ g of VEGF, 5 IGF-I~10 μ g, 5 EPO~10 μ g, surplus are injection
Use water.
2. a kind of fat according to claim 1 saves liquid, preparation method includes the following steps:
(1) DMEM in high glucose dehydrated medium 13.4g is poured into a container, with a small amount of water for injection by remaining medium in bag
It washes down, is incorporated to container；Inject water to 950ml, gentle agitation dissolution；
(2) 2.438 g sodium bicarbonates are added；
(3) gentle agitation dissolves, and injects water to 1L；
(4) pH value is adjusted to neutrality with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution；
(5) 2~10mg of insulin, 100~200mg of penicillin, 100~200mg of streptomysin, anphotericin are successively separately added into
2.5~5mg, 200 ~ 400mg of aspirin, 300~700mg of vitamin C, 7~12mg of resveratrol, icariin 2~
10mg, 20~40ug of hydrocortisone, 10~20 μ g of VEGF, 5 IGF-I~10 μ g, 5 EPO~10 μ g, mix well；
(6) degerming of 0.22um membrane filtration is made present invention fat and saves liquid, packing, freezen protective；It is placed in 0 when use in advance ~
8 DEG C of defrostings.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710828614.2A CN109497038A (en) | 2017-09-14 | 2017-09-14 | A kind of fat saves liquid and preparation method thereof |
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| CN201710828614.2A CN109497038A (en) | 2017-09-14 | 2017-09-14 | A kind of fat saves liquid and preparation method thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110663679A (en) * | 2019-10-24 | 2020-01-10 | 青岛瑞思德生物科技有限公司 | Placenta preserving fluid and preparation method thereof |
| CN113303326A (en) * | 2021-05-28 | 2021-08-27 | 吉林大学第一医院 | Organ preservation solution and liver in-vitro preservation method |
| CN113841688A (en) * | 2021-11-06 | 2021-12-28 | 马高辉 | Preservation solution for constructing mesenchymal stem cell bank and construction method of cell bank |
| CN113925049A (en) * | 2021-12-17 | 2022-01-14 | 广东乾晖生物科技有限公司 | Cell preservation solution for maintaining cell activity and preparation method and application thereof |
| CN114568422A (en) * | 2020-11-30 | 2022-06-03 | 北京瑷格干细胞科技有限公司 | Fat preservation liquid and application thereof |
| CN114651812A (en) * | 2022-02-22 | 2022-06-24 | 杨明 | Fat mesenchymal stem cell cryopreservation protective solution and cryopreservation method |
| WO2023061450A1 (en) * | 2021-10-13 | 2023-04-20 | 无锡赛比曼生物科技有限公司 | Adipose tissue transport preservation solution |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110663679A (en) * | 2019-10-24 | 2020-01-10 | 青岛瑞思德生物科技有限公司 | Placenta preserving fluid and preparation method thereof |
| CN114568422A (en) * | 2020-11-30 | 2022-06-03 | 北京瑷格干细胞科技有限公司 | Fat preservation liquid and application thereof |
| CN113303326A (en) * | 2021-05-28 | 2021-08-27 | 吉林大学第一医院 | Organ preservation solution and liver in-vitro preservation method |
| WO2023061450A1 (en) * | 2021-10-13 | 2023-04-20 | 无锡赛比曼生物科技有限公司 | Adipose tissue transport preservation solution |
| CN113841688A (en) * | 2021-11-06 | 2021-12-28 | 马高辉 | Preservation solution for constructing mesenchymal stem cell bank and construction method of cell bank |
| CN113925049A (en) * | 2021-12-17 | 2022-01-14 | 广东乾晖生物科技有限公司 | Cell preservation solution for maintaining cell activity and preparation method and application thereof |
| CN113925049B (en) * | 2021-12-17 | 2022-04-29 | 广东乾晖生物科技有限公司 | Cell preservation solution for maintaining cell activity and preparation method and application thereof |
| CN114651812A (en) * | 2022-02-22 | 2022-06-24 | 杨明 | Fat mesenchymal stem cell cryopreservation protective solution and cryopreservation method |
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