CN108812647A - Low irritability suitable for cat dog mescenchymal stem cell saves liquid and preparation method thereof - Google Patents
Low irritability suitable for cat dog mescenchymal stem cell saves liquid and preparation method thereof Download PDFInfo
- Publication number
- CN108812647A CN108812647A CN201810910338.9A CN201810910338A CN108812647A CN 108812647 A CN108812647 A CN 108812647A CN 201810910338 A CN201810910338 A CN 201810910338A CN 108812647 A CN108812647 A CN 108812647A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- mescenchymal stem
- cat
- cell
- cat dog
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
Abstract
The present invention provides a kind of low irritabilities being transported at room temperature suitable for cat dog mescenchymal stem cell to save liquid comprising distilled water and hydroxyethyl starch 130/0.4, glucose, citric acid, sodium citrate and sodium chloride.It is of the invention be suitable for the low irritability that cat dog mescenchymal stem cell is transported at room temperature save liquid at normal temperature can long period, long distance transportation mescenchymal stem cell, can guarantee cell viability and stability and can be entered with direct infusion and do not cause allergic reaction in vivo.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of low irritability guarantors suitable for cat dog mescenchymal stem cell
Liquid storage and preparation method thereof.
Background technique
Mescenchymal stem cell (Mesenchymal Stem Cells, MSC) is thin at soma from mesoderm development
Born of the same parents, it is most isolated prior in marrow, it is subsequently found it and is present in each tissue of body, such as fat, internal organ, muscle
And the postpartum such as placenta, amnion, umbilical cord discard tissue.Its ability with self-renewing and Multidirectional Differentiation, can be in vivo or in vitro
Induce differentiation into the cells such as fat, bone, cartilage, nerve.In recent years, with the rise of stem-cell therapy, MSC is applied to blood
The treatment of the diseases such as disease, skin trauma, lesions of liver and kidney, arthritis, myocarditis and cancer achieves satisfactory effect.
The transport of MSC is an important factor for influencing its clinical expansion and application and people want always the problem broken through.
Conventional MSC transport includes Cryopreservation transport and the adherent transport of room temperature, and Cryopreservation transportation cost is high and damages to cell
Hurt larger, containing the ingredients such as serum and DMSO in frozen stock solution, direct infusion is unable to after defrosting to internal；The adherent transportation resources of room temperature
Maintain the cell activity time short, and subsequent process steps are various, are unfavorable for a wide range of clinical expansion.In addition, if carrying out letter
Single cell infusion pre-treatment, the then it cannot be guaranteed that stability of cell and the activity of preservation, and cellular transport processes for a long time
In be easy agglomerating, conventional piping and druming cannot be such that it separates, and influence anaphase effect.
Summary of the invention
The purpose of the present invention is aiming at the above technical problems to be solved, provide it is a kind of at normal temperature can long period, remote
Distance transport mescenchymal stem cell and can guarantee the preservation liquid of cell viability and stability, the preservation liquid can direct infusion enter body
It is interior, it does not cause allergic reaction.
For this purpose, the present invention provides following technical schemes.
A kind of low irritability preservation liquid suitable for cat dog mescenchymal stem cell comprising distilled water, and based on described
The weight of distilled water be calculated as the glucose of 1.5~4.5% hydroxyethyl starch 130/0.4,0.5~1.5%, 0.014~
0.042% citric acid, 0.2~0.6% sodium citrate and 0.9% sodium chloride.
The present invention also provides the preparation method that the low irritability suitable for cat dog mescenchymal stem cell saves liquid, packets
It includes：
Hydroxyethyl starch, glucose, citric acid, sodium citrate, sodium chloride are weighed according to quantity and distilled water is added, it is high after dissolution
Pressure sterilizing saves liquid to get the low irritability.
Herein referred " high pressure sterilization " refers in closed autoclave sterilizer, and the saturated water of normal pressure is greater than using pressure
Steam kills microorganism, and steam sterilizing 15 is especially carried out in 102.97~137.30kPa of pressure, 121~126 DEG C of temperature
~30 minutes.
The present invention also provides a kind of methods for saving cat dog mescenchymal stem cell comprising：
The low irritability suitable for cat dog mescenchymal stem cell is added in cat dog mescenchymal stem cell to be saved to protect
In liquid storage.
Preferably, liquid is saved with the low irritability to adjust cat dog mescenchymal stem cell density to 1 × 106~5 × 106/
ml。
It is dry thin that liquid storage cat dog mesenchyma is saved using the low irritability suitable for cat dog mescenchymal stem cell of the invention
Born of the same parents compared with long distance transportation mescenchymal stem cell and can guarantee the vigor and stability of cell, and all the components safety in being formulated
It is nontoxic, clinically can direct infusion in vivo, adjuvant treatment.In addition, the preservation liquid is to the common high protein contents of previous people
It is improved, so that the sensitization of clinical direct infusion stem cell and storing liquid substantially reduces, safe without toxic side effect.
Detailed description of the invention
Fig. 1 shows P3 for dog fat mesenchymal stem cell adhered state.
Fig. 2 shows P3 for cat fat mesenchymal stem cell adhered state.
Fig. 3 is the adherent situation of cell that dog fat mesenchymal stem cell is seeded to after culture plate 6h.
Fig. 4 is the adherent situation of cell that cat fat mesenchymal stem cell is seeded to after culture plate 6h.
Specific embodiment
Combined with specific embodiments below, technical solution of the present invention is described in further detail, but the present invention is not limited to
Following embodiment.
As unspecified, related reagent can be bought by commercial sources below.For simplicity, part operation
The parameter, step and used instrument of operation are not described in detail, it should be understood that these are all well known to those skilled in the art and can
Repetition.
1. the culture of mescenchymal stem cell (by taking cat, dog fat mesenchymal stem cell as an example)
Healthy Dogs (cat) groin adipose tissue about 1cm is taken in aseptic operation table2In 50ml centrifuge tube, physiology salt is used
Water is shredded tissue block with dissecting scissors after cleaning bloodstain, and the collagenase type I that 5ml concentration is 1mg/ml is added and is placed in 37 DEG C of water-baths
Middle digestion 1h takes out 200 mesh net filtrations, and filtrate be centrifuged 5min abandoning supernatant at 1200rmp, with contain 10% fetal calf serum,
1% dual anti-, 1%L- glutamine DMEM culture solution is transferred in culture dish after precipitating is resuspended, and is placed in 37 DEG C, 5%CO2Culture
In case.It carries out changing liquid for the first time afterwards for 24 hours, washes away not adherent cell and impurity, changed a not good liquor every 2-3 days later.It sees daily
Examine cell state, when it is adherent to 80%-90% when can be passed on.
P3 is taken to carry out for cell into rouge Osteoblast Differentiation, and with the expression of its surface protein of identified by immunofluorescence CD34, CD44
Situation.
The results show that dog and cat fat mesenchymal stem cell express CD44 surface markers, CD34 surface markers are not expressed.
It as depicted in figs. 1 and 2, is P3 dog, cat fat mesenchymal stem cell adhered state (200 ×) respectively, picture is shown,
Cell is in fibrous adherent growth.
2. the preparation of cell-preservation liquid
It includes following component that the cell transport, which saves liquid,：Hydroxyethyl starch 130/0.4, glucose, citric acid, citric acid
Sodium, sodium chloride and distilled water, preparation steps are as follows：
Formula 1：Weigh hydroxyethyl starch (130/0.4) 3g, glucose 1g, citric acid 0.028g, sodium citrate 0.4g, chlorine
Change sodium 0.9g, distilled water 100ml.
Formula 2：Weigh hydroxyethyl starch (130/0.4) 1.5g, glucose 1.5g, citric acid 0.042g, sodium citrate
0.6g, sodium chloride 0.9g, distilled water 100ml.
Formula 3：Weigh hydroxyethyl starch (130/0.4) 4.5g, glucose 0.5g, citric acid 0.014g, sodium citrate
0.2g, sodium chloride 0.9g, distilled water 100ml.
High pressure sterilization after solid component is dissolved in distilled water by the above formula respectively, is placed in 4 DEG C of environment, saves
It is spare.
Above alleged " high pressure sterilization " refers in closed autoclave sterilizer, and the saturated water of normal pressure is greater than using pressure
Steam kills microorganism, and steam sterilizing 15 is especially carried out in 102.97~137.30kPa of pressure, 121~126 DEG C of temperature
~30 minutes.
3. the transport pre-treatment of mescenchymal stem cell
Take the adherent P3-P5 to 80%-90% for dog, cat fat mesenchymal stem cell, with 0.25% (mass concentration) pancreas
Digestion is terminated with the DMEM culture solution containing 10% (mass concentration) fetal calf serum after protease digestion, 1200rpm is centrifuged 5min, abandons
Cell is resuspended with physiological saline after supernatant, 1200rpm is centrifuged 5min again and abandons supernatant.It is then respectively adding what above-mentioned steps prepared
Cell-preservation liquid of the formula 1 to formula 3, adjustment cell density to 1 × 106~5 × 106/ ml takes 1ml cell suspension to 2ml EP
Guan Zhong, sealing orifice transport.
The dog that is prepared, cat mesenchyma stem cell suspension can directly carry out locally injecting or intravenous injection to dog, cat body
Interior, suspension needs the storage and transport under 4-25 DEG C of light protected environment, if environment temperature, which is more than 30 DEG C, may be used ice chest packaging fortune
Defeated MSC.
4. cytoactive detection
It is dispensed with the dog of the cell-preservation liquid resuspension of formula 1 to formula 3, cat mesenchyma stem cell suspension and is placed on 4 DEG C
It saves in refrigerator, is taken out for 24 hours, after 48h, 72h respectively at the, with viable count is counted after 0.4% Trypan Blue, living cells repels
Trypan blue, dead cell can then be dyed to blue, thus can calculate cell survival rate.Cell renewed vaccination is extremely trained after placing 72h
It supports in plate, the adherent situation of cell is observed after 6h.
Cell survival rate is as shown in the following Table 1.
Under the conditions of table 1 is transported at room temperature, cell survival rate calculated result after different time (n=3, results are averaged)
The adherent situation of cell after placement 72h after cell inoculation to culture plate 6h (by taking formula 1 as an example, is formulated 2 and formula 3
As a result similar) as shown in Figure 3 and Figure 4 (200 ×).Wherein, Fig. 3 is after dog fat mesenchymal stem cell is seeded to culture plate 6h
The adherent situation of cell.Fig. 4 is the adherent situation of cell that cat fat mesenchymal stem cell is seeded to after culture plate 6h.Such as Fig. 3 and Fig. 4
It has been shown that, cell well-grown.In addition, respectively that the dog being resuspended with the cell-preservation liquid of formula 1 to formula 3, cat mesenchyma is dry thin
Direct infusion enters cat, in dog body to born of the same parents' suspension respectively, and 10 practical infusion results show not cause allergic reaction.
Claims (4)
1. a kind of low irritability suitable for cat dog mescenchymal stem cell saves liquid, it is characterised in that including distilled water, Yi Jiji
In the weight of the distilled water be calculated as 1.5~4.5% hydroxyethyl starch 130/0.4,0.5~1.5% glucose, 0.014
~0.042% citric acid, 0.2~0.6% sodium citrate and 0.9% sodium chloride.
2. the preparation method of liquid is saved described in claim 1 suitable for the low irritability of cat dog mescenchymal stem cell comprising：
Hydroxyethyl starch, glucose, citric acid, sodium citrate, sodium chloride are weighed according to quantity and distilled water is added, and high pressure is gone out after dissolution
Bacterium saves liquid to get the low irritability suitable for cat dog mescenchymal stem cell.
3. a kind of method for saving cat dog mescenchymal stem cell comprising：
The low irritability suitable for cat dog mescenchymal stem cell is added in cat dog mescenchymal stem cell to be saved and saves liquid
In.
4. according to the method described in claim 3, cat dog mesenchyma is done carefully it is characterized in that, saving liquid with the low irritability
Born of the same parents' density is adjusted to 1 × 106~5 × 106/ml。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810910338.9A CN108812647A (en) | 2018-08-10 | 2018-08-10 | Low irritability suitable for cat dog mescenchymal stem cell saves liquid and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810910338.9A CN108812647A (en) | 2018-08-10 | 2018-08-10 | Low irritability suitable for cat dog mescenchymal stem cell saves liquid and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108812647A true CN108812647A (en) | 2018-11-16 |
Family
ID=64152932
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810910338.9A Pending CN108812647A (en) | 2018-08-10 | 2018-08-10 | Low irritability suitable for cat dog mescenchymal stem cell saves liquid and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108812647A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112426439A (en) * | 2020-11-17 | 2021-03-02 | 广东维赛生物科技有限公司 | Application of cat adipose-derived mesenchymal stem cells in preparation of preparation for treating cat acute kidney injury |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100167260A1 (en) * | 2006-01-19 | 2010-07-01 | Medestea Research & Production Spa | Use of N-Piperidine Derivative Compositions for Protecting Biological Systems |
| CN104857022A (en) * | 2015-05-21 | 2015-08-26 | 北京青藤谷禧干细胞科技研究院有限公司 | MSC (mesenchymal stem cell) injection as well as preparation and application thereof |
| CN107912424A (en) * | 2017-12-01 | 2018-04-17 | 佛山科学技术学院 | A kind of special room-temperature extender of dog mescenchymal stem cell and application |
-
2018
- 2018-08-10 CN CN201810910338.9A patent/CN108812647A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100167260A1 (en) * | 2006-01-19 | 2010-07-01 | Medestea Research & Production Spa | Use of N-Piperidine Derivative Compositions for Protecting Biological Systems |
| CN104857022A (en) * | 2015-05-21 | 2015-08-26 | 北京青藤谷禧干细胞科技研究院有限公司 | MSC (mesenchymal stem cell) injection as well as preparation and application thereof |
| CN107912424A (en) * | 2017-12-01 | 2018-04-17 | 佛山科学技术学院 | A kind of special room-temperature extender of dog mescenchymal stem cell and application |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112426439A (en) * | 2020-11-17 | 2021-03-02 | 广东维赛生物科技有限公司 | Application of cat adipose-derived mesenchymal stem cells in preparation of preparation for treating cat acute kidney injury |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101490851B1 (en) | Aqueous moisturizers and lubricants and uses thereof | |
| CN106538512B (en) | A kind of active stem cell gel preparation of holding freeze-stored cell and its application | |
| JP4421681B2 (en) | Methods and compositions for improving survival and function of germ cells and embryos | |
| JP5230042B2 (en) | Preservatives for animal cells or organs and methods for their preservation. | |
| CN110050782A (en) | A kind of stem cell cryopreserving liquid and preparation method thereof and cryopreservation methods | |
| EP3517118B1 (en) | Sperm activator and uses thereof | |
| CA3046566A1 (en) | Post-partum mammalian placental stem cells for use in the treatment of neurological or renal diseases and disorders | |
| CN110959606B (en) | Immune effector cell cryopreservation liquid and application thereof | |
| EP1094703A2 (en) | Improved methods for storing neural cells | |
| CN107912424A (en) | A kind of special room-temperature extender of dog mescenchymal stem cell and application | |
| CN107106488A (en) | Purposes of the trehalose in cell suspension | |
| JP2022501322A (en) | A method for obtaining an enriched population of functional mesenchymal stem cells, the cells thus obtained, and a composition comprising the cells. | |
| CN108056095A (en) | A kind of fat mesenchymal stem cell transport protection liquid and its application | |
| CN109825469A (en) | A kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to Chondrocyte Differentiation | |
| Liu et al. | Effect of sodium alginate on mouse ovary vitrification | |
| CN109619088A (en) | A kind of preservation liquid of fat mesenchymal stem cell | |
| CN109832261A (en) | A kind of cells frozen storing liquid and its application | |
| JPWO2005115141A1 (en) | Cell / tissue / organ preservation solution using rare sugar and preservation method using the solution | |
| CN113973805B (en) | Cell cryopreservation kit and use method thereof | |
| JP2003267801A (en) | Composition for preservative and preservative of cell or organ of animal containing the same composition | |
| CN105055450A (en) | Applications of human endometrium stem cells in preparation of drugs for premature ovarian failure treatment | |
| CN108812647A (en) | Low irritability suitable for cat dog mescenchymal stem cell saves liquid and preparation method thereof | |
| CN106701682A (en) | Method for separating hematopoietic stem cells from umbilical cord blood and amplifying CD34 positive cells | |
| CN110520138A (en) | What is obtained from wharton's jelly is used to treat pyemic mescenchymal stem cell | |
| El-Shewy et al. | Polyvinyl pyrrolidone: a novel cryoprotectant in islet cell cryopreservation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181116 |