CN107912424A - A kind of special room-temperature extender of dog mescenchymal stem cell and application - Google Patents
A kind of special room-temperature extender of dog mescenchymal stem cell and application Download PDFInfo
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- CN107912424A CN107912424A CN201711251147.8A CN201711251147A CN107912424A CN 107912424 A CN107912424 A CN 107912424A CN 201711251147 A CN201711251147 A CN 201711251147A CN 107912424 A CN107912424 A CN 107912424A
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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
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Abstract
The present invention provides a kind of special room-temperature extender of dog mescenchymal stem cell, including following component：Dog blood albumin, physiological saline, glucose, citric acid, sodium chloride.The application process of the above-mentioned special room-temperature extender of dog mescenchymal stem cell is additionally provided, including：It will be added by the dog mescenchymal stem cell of transport pre-treatment in the special room-temperature extender of dog mescenchymal stem cell, adjustment cell density to 5 × 105~6/ ml, takes 1ml cell suspensions into 2ml EP pipes, sealing.The special room-temperature extender of dog mescenchymal stem cell is easily prepared, and manufacturing cost is relatively low, has good preservation dog mescenchymal stem cell effect at normal temperatures, effectively prevents that dog mescenchymal stem cell is dead or agglomerating.
Description
Technical field
The present invention relates to technical field of bioengineering.
Background technology
Mescenchymal stem cell (Mesenchymal Stem Cells, MSC) is developed by mesoderm, is present in body
Adult stem cell in each tissue, its ability with self-renewing and Multidirectional Differentiation.In recent years, with stem-cell therapy
Rise, MSC is applied to the treatment of the diseases such as hematologic disease, skin trauma, lesions of liver and kidney, arthritis, myocarditis and cancer, takes
Obtained gratifying effect.
The transport of MSC be influence its it is clinical it is general with application an important factor for, and people want the problem broken through always.Often
The MSC transports of rule include ultralow temperature transport and the adherent transport of room temperature, and not only cost of transportation is high for both approaches, and subsequent operation
Step is various, is unfavorable for a wide range of clinical expansion.In addition, if carry out simple cell infusion pre-treatment, then it is it cannot be guaranteed that thin
The stability of born of the same parents and the activity preserved for a long time, and it is easily agglomerating in cellular transport processes, and conventional piping and druming cannot separate it,
Influence anaphase effect.
The content of the invention
To solve the high cost of above-mentioned technology, the shortcomings that processing method is complicated and deficiency, the present invention provides one kind in room temperature
Down can the long period, long distance transportation mescenchymal stem cell preservation liquid, particularly the mescenchymal stem cell in dog source is protected
There are in the preservation liquid, easy to storage and transport MSC, and ensure the vigor and stability of cell.
The present invention is achieved by the following technical solutions：
A kind of special room-temperature extender of dog mescenchymal stem cell, including following component：Dog blood albumin, physiological saline, Portugal
Grape sugar, citric acid, sodium chloride.Specifically by solution A and solution B with 1:1 ratio forms, and the solution A is per in 100ml aqueous solutions
Include glucose 2.05g, citric acid 0.055g, sodium chloride 0.42g；The solution B is to take dog blood albumin to be dissolved in physiological saline
10% dog albumin solution being formulated.The solution A is preserved through autoclaving and 4 DEG C.The dog mescenchymal stem cell
After special room-temperature extender prepares, using preceding with 4 DEG C of preservations.
Present invention also offers the application process of the above-mentioned special room-temperature extender of dog mescenchymal stem cell, including：It will pass through
The dog mescenchymal stem cell for transporting pre-treatment is added in the special room-temperature extender of dog mescenchymal stem cell, adjusts cell density
To 5 × 105~6/ ml, takes 1ml cell suspensions into 2ml EP pipes, sealing.
Wherein, the transport pre-treatment includes step：A turn generation is taken to be filled between P3~P5 generations, the adherent dog to 80~90%
Matter stem cell, after being terminated with pancreatin digestion, 1200rpm centrifugation 5min, abandon after supernatant and cell, 1200rpm are resuspended with physiological saline
Centrifugation 5min abandons supernatant again.
The beneficial effects of the invention are as follows：The special room-temperature extender of dog mescenchymal stem cell is easily prepared, and manufacturing cost
It is relatively low, there is good preservation dog mescenchymal stem cell effect at normal temperatures, effectively prevent that dog mescenchymal stem cell is dead or agglomerating.
Brief description of the drawings
Fig. 1 is P3 for dog mescenchymal stem cell adhered state figure；
Fig. 2 is that dog mescenchymal stem cell is seeded to the adherent situation of culture plate after placing 72h described in embodiment 4.
Embodiment
The present invention is described in further detail below by embodiment combination attached drawing.But those skilled in the art
It will be understood that the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment
Particular technique or condition person, carry out according to the described technology of document in the art or condition or according to product description.
Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
Embodiment 1：The culture of dog mescenchymal stem cell.
By taking dog fat mesenchymal stem cell as an example, Healthy Dogs groin adipose tissue about 1cm is taken in aseptic operation table2In
In 50ml centrifuge tubes, tissue block is shredded with dissecting scissors after cleaning bloodstain with physiological saline, adds 5ml 1mg/ml Type I collagen enzymes
It is placed in 37 DEG C of water-baths and digests 1h, 5min is centrifuged under 1200rmp and is abandoned supernatant with 200 mesh sieve net filtrations, filtrate after taking-up, uses
It is transferred in culture dish, is placed in after precipitation is resuspended containing 10% hyclone, 1% dual anti-, 1%L- glutamine DMEM nutrient solutions
37 DEG C, 5%CO2In incubator.Carry out changing liquid for the first time after 24h, not adherent cell and impurity are washed away, later every 2-3 days
Change a not good liquor.Daily observation cell state, when it is adherent to 80%-90% when can be passed on.Take P3 for cell carry out into fat into
Bone breaks up, as shown in Figure 1, and with the expression of its surface protein of identified by immunofluorescence CD34, CD44, CD90.
Embodiment 2：The preparation of special room-temperature extender.
Solution A：Weigh glucose 2.05g, citric acid 0.055g, sodium chloride 0.42g and add distilled water to 100ml, after dissolving
Autoclaving, is placed in 4 DEG C of environment, saves backup.
Solution B：Weigh 1g dog blood albumin and be dissolved in 10ml physiological saline and be configured to 10% dog albumin solution.
By 1：1 ratio takes above-mentioned solution A and solution B respectively, and mixing is placed on spare in 4 DEG C of refrigerators.
Embodiment 3：The transport pre-treatment of MSC.
In the dog mescenchymal stem cell that Example 1 is cultivated, a turn generation, fill between P3~P5 generations, the adherent dog to 80~90%
Matter stem cell, after being terminated with pancreatin digestion, 1200rpm centrifugation 5min, abandon after supernatant and cell, 1200rpm are resuspended with physiological saline
Centrifugation 5min abandons supernatant again.Add in the special room-temperature extender of dog mescenchymal stem cell made from embodiment 2, adjustment cell is close
Spend to 5 × 105~6/ ml, takes 1ml cell suspensions into 2ml EP pipes, sealing.
The dog mesenchyma stem cell suspension being prepared can directly carry out local injection or intravenous injection is arrived in dog body, suspension
The storage and transport under 4-25 DEG C of light protected environment are needed, after preparation in 48h effectively.
Embodiment 4：Dog mescenchymal stem cell Activity determination.
EP pipes containing dog mescenchymal stem cell and special room-temperature extender prepared by embodiment 3 are divided into 3 groups, put respectively
Preserve under 4 DEG C, 25 DEG C and 37 DEG C environment, taken out after 4h, 24h, 48h, 72h, with being counted after 0.4% Trypan Blue
Viable count, living cells repel trypan blue, and dead cell can then be dyed to blueness, thus can calculate cell survival rate.Survival rate is such as
Shown in table 1.
Under 1 condition of different temperatures of table, cell survival rate result of calculation after different time is placed
After (25 DEG C) placement 72h of room temperature cell renewed vaccination is observed into cell attachment situation such as Fig. 2 into culture plate after 6h
It is shown.
Claims (6)
1. a kind of special room-temperature extender of dog mescenchymal stem cell, it is characterised in that including following component：Dog blood albumin, physiology
Brine, glucose, citric acid, sodium chloride.
2. the special room-temperature extender of dog mescenchymal stem cell according to claim 1, it is characterised in that by solution A and molten
Liquid B is with 1:1 ratio forms, and the solution A is comprising glucose 2.05g, citric acid 0.055g, chlorination in every 100ml aqueous solutions
Sodium 0.42g；The solution B is to take dog blood albumin to be dissolved in the 10% dog albumin solution that normal saline forms.
3. the special room-temperature extender of dog mescenchymal stem cell according to claim 2, it is characterised in that the solution A warp
Autoclaving and 4 DEG C of preservations.
4. the special room-temperature extender of dog mescenchymal stem cell according to claim 2, it is characterised in that the dog mesenchyma
The special room-temperature extender of stem cell is using preceding with 4 DEG C of preservations.
5. the application process of the special room-temperature extender of dog mescenchymal stem cell, including：By the dog mesenchyma by transport pre-treatment
Stem cell is added in the special room-temperature extender of dog mescenchymal stem cell as described in any one of Claims 1 to 4, adjusts cell density
To 5 × 105~6/ ml, takes 1ml cell suspensions into 2ml EP pipes, sealing.
6. application process according to claim 5, it is characterised in that the transport pre-treatment includes step：Take and turn generation extremely
It is P3~P5 generations, adherent to 80~90% dog mescenchymal stem cell, with pancreatin digestion terminate after, 1200rpm centrifugation 5min, abandon
Cell is resuspended with physiological saline after supernatant, 1200rpm centrifuges 5min and abandons supernatant again.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108812647A (en) * | 2018-08-10 | 2018-11-16 | 广东维赛生物科技有限公司 | Low irritability suitable for cat dog mescenchymal stem cell saves liquid and preparation method thereof |
| CN109402178A (en) * | 2018-11-16 | 2019-03-01 | 佛山科学技术学院 | A kind of method and application that spermatogonial stem cells into mouse efficiently reprograms |
| CN113498779A (en) * | 2021-04-22 | 2021-10-15 | 重庆医科大学附属儿童医院 | Reagent and method for transporting cells |
| CN115590018A (en) * | 2022-11-21 | 2023-01-13 | 佛山科学技术学院（Cn） | Clinical mesenchymal stem cell refrigerating preservation solution for dogs and application method thereof |
| CN116808076A (en) * | 2023-08-25 | 2023-09-29 | 北京臻宠生物科技有限公司 | Application method of mesenchymal stem cell repair factor in canine wound treatment |
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| CN106540244A (en) * | 2016-12-06 | 2017-03-29 | 佛山科学技术学院 | A kind of dog mesenchymal stem cell injection and its preparation method and application |
| CN106554940A (en) * | 2015-09-24 | 2017-04-05 | 苏州爱乐桢医疗器械有限公司 | A kind of fat mesenchymal stem cell transport protection liquid |
| CN106982821A (en) * | 2017-05-22 | 2017-07-28 | 安徽瑞杰赛尔生物科技有限公司 | Umbilical cord mesenchymal stem cells clinic freezes protection liquid composition and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108812647A (en) * | 2018-08-10 | 2018-11-16 | 广东维赛生物科技有限公司 | Low irritability suitable for cat dog mescenchymal stem cell saves liquid and preparation method thereof |
| CN109402178A (en) * | 2018-11-16 | 2019-03-01 | 佛山科学技术学院 | A kind of method and application that spermatogonial stem cells into mouse efficiently reprograms |
| CN109402178B (en) * | 2018-11-16 | 2021-08-03 | 佛山科学技术学院 | Efficient reprogramming method and application of mouse spermatogonial stem cells |
| CN113498779A (en) * | 2021-04-22 | 2021-10-15 | 重庆医科大学附属儿童医院 | Reagent and method for transporting cells |
| CN115590018A (en) * | 2022-11-21 | 2023-01-13 | 佛山科学技术学院（Cn） | Clinical mesenchymal stem cell refrigerating preservation solution for dogs and application method thereof |
| CN116808076A (en) * | 2023-08-25 | 2023-09-29 | 北京臻宠生物科技有限公司 | Application method of mesenchymal stem cell repair factor in canine wound treatment |
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Application publication date: 20180417 |