CN108849855A - A kind of adipose tissue Plantlet in vitro liquid and preparation method thereof - Google Patents
A kind of adipose tissue Plantlet in vitro liquid and preparation method thereof Download PDFInfo
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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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- 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 adipose tissue Plantlet in vitro liquid and preparation method thereof.The preservation liquid comprises the following components in parts by weight：0.5~1.5 part of autologous platelet lysate, 0~5 part of Sodium Hyaluronate, 1~5 part of human serum albumin, 0~1 part of Vc, 0.1~0.3 part of trehalose, 10~20 parts of temperature-sensitive hydrogel, 0.3~1.5 part of antibacterials and 80~90 parts of phosphate buffer；Preparation method is：Aseptically, each component is mixed by formula, adipose tissue Plantlet in vitro liquid is prepared.The preservation liquid that the method for the present invention is prepared can be such that adipose tissue is uniformly dispersed to and save in liquid, come into full contact with adipose tissue with liquid is saved, be conducive to the long term storage of adipose tissue.
Description
Technical field
The invention belongs to cell engineering fields, and in particular to a kind of adipose tissue Plantlet in vitro liquid and its preparation side
Method.
Background technique
Stem cell (stem cell) is a kind of multipotential cell with the of self-replication capacity (self-renewing), with
Deepen continuously to stem-cell research, the stem cells of more and more separate sources becomes the research hotspot of each research institution, such as
Bone marrow mescenchymal stem cell, placenta source mescenchymal stem cell, dental pulp source mescenchymal stem cell, adipose tissue-derived mesenchymal stem cells
Deng.
With the development of adipose tissue engineering, adipose-derived stem cell (and at fat source vascular-stromal cells) is more and more
It is taken seriously, which are mainly applied to the fields such as medical and beauty treatment.It is latent that Zuk in 2001 etc. is found to have multinomial differentiation in adipose tissue
Since energy, fibroblastic stem cell, the characteristics such as multi-lineage potential of fat stem cell are expected to as organization work
One of " seed cell ".
Adipose tissue rich reserves and acquisition simplicity, the fat stem cell (adipose obtained from adipose tissue
Tissue-derived stem cells, ADSCs) in different induced environments can at rouge, skeletonization, at cartilage direction
Differentiation, and a variety of growth factors and cell factor can be secreted, it is expected to the ideal stem cell that planning is summarized as in clinical application
Source provides new thinking for the application of regenerative medicine field.
The triglyceride that fat is made of glycerol and fatty acid, wherein the molecule of glycerol is fairly simple, and fatty acid
Type and length it is but not identical.Fatty acid divides three categories：Saturated fatty acid, monounsaturated fatty acids, polyunsaturated fatty acid.
Fat soluble is not dissolved in water in most organic solvents.It is the glycerolipid C of one or more kinds of fatty acid3H5(OOCR)3,
To extend the in vitro storage deadline of adipose tissue and guaranteeing fat stem cell success rate of extracting, in vitro adipose tissue need to be stored in
Adipose tissue saves in liquid, provides nutrition supply for adipose tissue.
If transport saves isolated adipose tissue under normal temperature conditions for a long time, it is easy to damage isolated adipose tissue, and
Fat is not soluble in water, and density is lighter, saves the adipose tissue in liquid and can be suspended in and saves in liquid, so that adipose tissue can not be with
It saves liquid to come into full contact with, is unable to satisfy the needs that adipose tissue transport saves.
Summary of the invention
For above-mentioned deficiency in the prior art, the present invention provides a kind of adipose tissue Plantlet in vitro liquid, can effectively solve
The problem of in Plantlet in vitro, being suspended in for appearance saves on liquid adipose tissue, can not come into full contact with preservation liquid.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is：
A kind of adipose tissue Plantlet in vitro liquid, comprises the following components in parts by weight：
0.5~1.5 part of autologous platelet lysate, 0~5 part of Sodium Hyaluronate, 1~5 part of human serum albumin, Vc0~1
Part, 0.1~0.3 part of trehalose, 10~20 parts of temperature-sensitive hydrogel, 0.3~1.5 part of antibacterials and phosphate buffer 80
~90 parts.
Further, it comprises the following components in parts by weight：
1 part of autologous platelet lysate, 2 parts of Sodium Hyaluronate, 3 parts of human serum albumin, 1 part of Vc, 0.2 part of trehalose, temperature
12 parts of quick hydrogel, 0.82 part of antibacterials and 84 parts of phosphate buffer.
Further, the preparation method of temperature-sensitive hydrogel is as follows：
Polyacrylamide, methacrylic acid, acrylic acid and crosslinking agent are mixed, initiator is added, stirs evenly, it is quiet
After setting 10~15h of reaction, takes out reaction product and wash 3~5 times；Wherein, polyacrylamide, methacrylic acid, acrylic acid, crosslinking
The weight ratio of agent and initiator is 5~10:0.5~1:0.5~1:0.15~0.3:0.5~1.
Further, the weight ratio of polyacrylamide, methacrylic acid, acrylic acid, crosslinking agent and initiator is 8:1:
0.6:0.15:1。
Further, crosslinking agent is chitosan.
Further, initiator is ammonium persulfate, potassium peroxydisulfate, lauroyl peroxide or sodium peroxydisulfate.
Further, antibacterials include penicillin, streptomysin and anphotericin；Wherein, penicillin, streptomysin and both sexes
The weight ratio of mycin B is：0.2~0.6:0.1~0.5:0.01~0.05.
Further, the weight ratio of penicillin, streptomysin and amphotericin B is：0.5:0.3:0.02.
Further, phosphate buffer pH value is 7.2~7.6.
The preparation method of above-mentioned adipose tissue Plantlet in vitro liquid, includes the following steps：
Aseptically, each component in claim 1 is mixed by formula, adipose tissue Plantlet in vitro is prepared
Liquid.
Beneficial effects of the present invention are：
1, temperature-sensitive hydrogel is that have three-dimensional by what is formed after polyacrylamide, methacrylic acid and acrylic acid cross-linked polymeric
According to variation of ambient temperature volume phase transition phenomena can occur for the hydrogel of network structure；When temperature is higher than minimum critical
When solution temperature (LCST), the imprinted sites on macromolecular chain are close to each other, in swelling memory loss state, i.e., are at normal temperature in liquid
State；When temperature is lower than lowest critical solution temperature (LCST), the imprinted sites on macromolecular chain are located remotely from each other, and are remembered in shrinking
Recall state, i.e., is at low temperature in solid-state；As a result, by adding the temperature-sensitive hydrogel in saving liquid, then at normal temperature need to
The adipose tissue to be saved is added into the preservation liquid, and concussion mixes, and then saves at 0~8 DEG C, meanwhile, in the hydrogel
It is shaken frequently before not solidifying, so that adipose tissue is uniformly distributed in and save in liquid, and make the preservation liquid in transportational process
In be in gel state, with guarantee adipose tissue can with save liquid come into full contact with, conducive to the long-term storage of adipose tissue and cell
It deposits.
2, autologous platelet lysate be extracted from fatty donor self-blood it is isolated, including a large amount of
Porcine HGF, transforming growth factor, vascular endothelial growth factor etc. can provide for adipose tissue, fat cell
Better nutrition supply environment；The ascorbic addition of ascorbic acid-can help fat cell to maintain various peroxidase
Activity prevents oxygen radical from causing to damage to cell membrane and organelle, plays a key effect to adipose tissue is saved.
3, human serum albumin can be used as stabilizer and protective agent, and be the important component of cell culture medium, have relatively strong
Protective effect, the growth of cell can be promoted, extend the time of cell survival；Increase blood volume and maintain osmotic pressure, mainly
The dynamic equilibrium of moisture between tissue and blood vessel is adjusted, and can be used as nitrogen source is tissue with nutrient.
4, trehalose (trehalose) is the irreducibility disaccharide being widely present in a kind of nature, is had no toxic side effect, and is changed
It is sufficiently stable to learn property, in addition to the general characteristic with oligosaccharide, there are also unique biological characteristics, can protect under harsh environment
The tissue of probationer nurse object and the function of macromolecular and activity；1) stablize the structure of biomembrane and protein；2) protection organism is exempted from
Heated and dry damage；3) damage of oxidative stress is protected organisms from；Oxygen radical can make the ammonia in cell protein
Base acid is impaired and leads to the damage to cell, and the trehalose of cell middle and high concentration can prevent this damage, this shows trehalose
Has the function of oxygen free radical scavenger；4) trehalose can protect mammalian cell from the damage of anoxic.
5, the preservation liquid that the method for the present invention is prepared can not only enable adipose tissue adequately to contact with preservation liquid,
The holding time of adipose tissue can also effectively be extended.
Detailed description of the invention
Fig. 1 is the flow cytometer detection figure of CD105 and CD45 expression quantity；
Fig. 2 is the flow cytometer detection figure of CD90 expression quantity；
Fig. 3 is the flow cytometer detection figure of CD19 expression quantity；
Fig. 4 is the flow cytometer detection figure of CD11b expression quantity；
Fig. 5 is the flow cytometer detection figure of CD34 expression quantity；
Fig. 6 is the flow cytometer detection figure of CD73 and HLA-DR expression quantity；
Fig. 7 is thermo-sensitive gel gelation temperature test map.
Specific embodiment
A specific embodiment of the invention is described below, in order to facilitate understanding by those skilled in the art this hair
It is bright, it should be apparent that the present invention is not limited to the ranges of specific embodiment, for those skilled in the art,
As long as various change is in the spirit and scope of the present invention that the attached claims limit and determine, these variations are aobvious and easy
See, all are using the innovation and creation of present inventive concept in the column of protection.
Embodiment 1
A kind of adipose tissue Plantlet in vitro liquid contains autologous platelet lysate 1mL, transparent in the every 100mL of the preservation liquid
Matter acid sodium 2g, human serum albumin 3mL, Vc 1g, trehalose 0.2g, temperature-sensitive hydrogel 12mL, penicillin 0.5g, streptomysin 0.3g,
Amphotericin B 0.02g, remaining solvent medium are the phosphate buffer that pH value is 7.4.
Wherein, the preparation method of temperature-sensitive hydrogel is：By polyacrylamide, methacrylic acid, acrylic acid and chitosan
Mixing, is then added ammonium persulfate, stirs evenly, and after standing reaction 15h, washs 3 secondary response products, obtains temperature-sensitive hydrogel；Its
In, polyacrylamide, methacrylic acid, acrylic acid, chitosan and ammonium persulfate weight ratio be 8:1:0.6:0.15:1.
The preparation method of the preservation liquid is：
Aseptically, above-mentioned each ingredient is uniformly mixed, adipose tissue Plantlet in vitro liquid is prepared.
The method for saving adipose tissue using the preservation liquid is as follows：
By adipose tissue and liquid is saved according to 0.5:1 solid-liquid ratio mixes under normal temperature state, and concussion mixes, and is subsequently placed in
It is saved at 0~8 DEG C, meanwhile, it is shaken frequently before the preservation liquid does not solidify, until the preservation liquid solidifies completely.
Embodiment 2
A kind of adipose tissue Plantlet in vitro liquid contains autologous platelet lysate 0.5mL, people in the every 100mL of the preservation liquid
Blood albumin 1mL, Vc 0.5g, trehalose 0.3g, temperature-sensitive hydrogel 20mL, penicillin 0.6g, streptomysin 0.5g, amphotericin B
0.05g, remaining solvent medium are the phosphate buffer that pH value is 7.6.
Wherein, the preparation method of temperature-sensitive hydrogel is：By polyacrylamide, methacrylic acid, acrylic acid and chitosan
Mixing, is then added sodium peroxydisulfate, stirs evenly, and after standing reaction 15h, washs 3 secondary response products, obtains temperature-sensitive hydrogel；Its
In, polyacrylamide, methacrylic acid, acrylic acid, chitosan and sodium peroxydisulfate weight ratio be 10:0.5:0.8:0.2:
0.6。
The preparation method of the preservation liquid is：
Aseptically, above-mentioned each ingredient is uniformly mixed, adipose tissue Plantlet in vitro liquid is prepared.
The method for saving adipose tissue using the preservation liquid is as follows：
By adipose tissue and liquid is saved according to 0.5:1 solid-liquid ratio mixes under normal temperature state, and concussion mixes, and is subsequently placed in
It is saved at 0~8 DEG C, meanwhile, it is shaken frequently before the preservation liquid does not solidify, until the preservation liquid solidifies completely.
Embodiment 3
A kind of adipose tissue Plantlet in vitro liquid, in the every 100mL of the preservation liquid containing autologous platelet lysate 1.5mL, thoroughly
Bright matter acid sodium 5g, human serum albumin 5mL, trehalose 0.1g, temperature-sensitive hydrogel 10mL, penicillin 0.2g, streptomysin 0.1g, both sexes
Mycin B 0.01g, remaining solvent medium are the phosphate buffer that pH value is 7.2.
Wherein, the preparation method of temperature-sensitive hydrogel is：By polyacrylamide, methacrylic acid, acrylic acid and chitosan
Mixing, is then added lauroyl peroxide, stirs evenly, and after standing reaction 15h, washs 3 secondary response products, obtains temperature sensitive water-setting
Glue；Wherein, the weight ratio of polyacrylamide, methacrylic acid, acrylic acid, chitosan and lauroyl peroxide is 6:0.8:1:
0.3:1。
The preparation method of the preservation liquid is：
Aseptically, above-mentioned each ingredient is uniformly mixed, adipose tissue Plantlet in vitro liquid is prepared.
The method for saving adipose tissue using the preservation liquid is as follows：
By adipose tissue and liquid is saved according to 0.5:1 solid-liquid ratio mixes under normal temperature state, and concussion mixes, and is subsequently placed in
It is saved at 0~8 DEG C, meanwhile, it is shaken frequently before the preservation liquid does not solidify, until the preservation liquid solidifies completely.
Experimental example
1, thermo-sensitive gel gelation temperature detects
In under room temperature, the thermo-sensitive gel being prepared in embodiment 1 is placed on magnetic stirring apparatus, wherein stirring
Sub- size be 0.6mm × 1.5mm, stirring rate 200r/min, then respectively heating cooling, until stirrer stop operating for
Only, and detect thermo-sensitive gel colloid temperature, gelation temperature test map are as shown in Figure 7 at this time.
As shown in Figure 7, when being more than or equal to 25 DEG C, stirring rate of the stirrer in thermo-sensitive gel is not any change, and
With the reduction of temperature, the stirring rate of stirrer is gradually decreased, until stopping completely at 3~5 DEG C, showing the temperature as a result,
Quick gel is in a liquid state when temperature is greater than room temperature, is in solid-state when temperature is reduced to 3~5 DEG C.
2, the acquisition of adipose tissue
Donor requires the pathogenic microorganism examination result negative, specific as follows：HBV, HCV, HIV, HTLV, EBV, TP, fat
Source is not limited only to waist and belly, buttocks, huckle etc., and the mode of liposuction is taken to be acquired, and is divided into two parts, a copy of it
It is sent to laboratory immediately and extracts culture, another is put into the preservation liquid that the embodiment of the present invention 1 is prepared, in 2-8 DEG C
Culture is extracted after saving in environment 3 weeks.
3, the upper-layer fat tissue after taking room temperature stratification, after being mixed well with isometric sterile saline in
2000rpm/min, 5min, removal oil droplet, white fascia, haemocyte are centrifuged under the conditions of 20 DEG C.
4, the adipose tissue after 3 steps of repetition must clean.
5, adipose tissue is inoculated in culture bottle, every bottle of 2-3ml；Be placed in 37 DEG C, the saturation of 5% gas concentration lwevel it is wet
It is cultivated in degree incubator.
6, cell adhered state is observed, changes liquid within about every 3-5 days, carries out passage processing when attached cell grows to 60-80%,
Adipose tissue is discarded, and is cleaned 1-2 times with sterile saline, 0.25% trypsase containing EDTA restored to room temperature is added
Digest 2-3min.
7, according to 1~1.5x104A/cm2Inoculative proportion carry out passage amplification cultivation.
Fresh fat and the cell state for saving the fat after liquid stores being prepared through the embodiment of the present invention 1 are carried out
Detection, testing result are shown in Table 1.
The cell concentration and motility rate that 1 adipose tissue of table extracts
Testing result is as shown in table 1, and adipose tissue is protected by the preservation liquid that 1 the method for the embodiment of the present invention is prepared
After depositing 3 weeks, the extraction culture of fat stem cell is carried out, cell quantity and motility rate and fresh rouge after obtained primary and amplification
The fat stem cell of fat tissue extraction is almost the same, illustrates that preservation liquid that the method for the present invention is prepared can effectively ensure that and mentions
The activity of the adipose tissue taken out.
Result is shown in Table 2 and Fig. 1 respectively to be identified for the immunophenotype of cell to the P1 that the method for the present invention extracts again
~Fig. 6；Wherein, Fig. 1 is the flow cytometer detection figure of CD105 and CD45 expression quantity；Fig. 2 is the flow cytometer detection figure of CD90 expression quantity；Fig. 3
For the flow cytometer detection figure of CD19 expression quantity；Fig. 4 is the flow cytometer detection figure of CD11b expression quantity；Fig. 5 is that the streaming of CD34 expression quantity is examined
Mapping；Fig. 6 is the flow cytometer detection figure of CD73 expression quantity；
The expression quantity of immunological marker object in 2 fat stem cell of table
According to FIG. 1 to FIG. 6 and the detection data of table 2 it is found that the extracted institute of adipose tissue saved by this preservation liquid
Stem cell has a typical mescenchymal stem cell surface marker feature, the marker of high expression quantity is CD73, CD90, CD105,
And positive expression rate is all larger than 99%, the marker of low expression amount is CD11b, CD19, CD34, CD45, HLA-DR, feminine gender expression
Rate is respectively less than 2%.
Claims (10)
1. a kind of adipose tissue Plantlet in vitro liquid, which is characterized in that comprise the following components in parts by weight：
0.5~1.5 part of autologous platelet lysate, 0~5 part of Sodium Hyaluronate, 1~5 part of human serum albumin, Vc0~1 part, sea
0.1~0.3 part of algae sugar, 10~20 parts of temperature-sensitive hydrogel, 0.3~1.5 part of antibacterials and phosphate buffer 80~90
Part.
2. adipose tissue Plantlet in vitro liquid according to claim 1, which is characterized in that comprise the following components in parts by weight：
1 part of autologous platelet lysate, 2 parts of Sodium Hyaluronate, 3 parts of human serum albumin, 1 part of Vc, 0.2 part of trehalose, temperature sensitive water
12 parts of gel, 0.82 part of antibacterials and 84 parts of phosphate buffer.
3. adipose tissue Plantlet in vitro liquid according to claim 1 or 2, which is characterized in that the system of the temperature-sensitive hydrogel
Preparation Method is as follows：
Polyacrylamide, methacrylic acid, acrylic acid and crosslinking agent are mixed, initiator is added, stirs evenly, is stood anti-
After answering 10~15h, takes out reaction product and wash 3~5 times；Wherein, polyacrylamide, methacrylic acid, acrylic acid, crosslinking agent with
And the weight ratio of initiator is 5~10:0.5~1:0.5~1:0.15~0.3:0.5~1.
4. adipose tissue Plantlet in vitro liquid according to claim 3, which is characterized in that the polyacrylamide, methyl-prop
Olefin(e) acid, acrylic acid, crosslinking agent and initiator weight ratio be 8:1:0.6:0.15:1.
5. adipose tissue Plantlet in vitro liquid according to claim 3 or 4, which is characterized in that the crosslinking agent is chitosan.
6. adipose tissue Plantlet in vitro liquid according to claim 3 or 4, which is characterized in that the initiator is persulfuric acid
Ammonium, potassium peroxydisulfate, lauroyl peroxide or sodium peroxydisulfate.
7. adipose tissue Plantlet in vitro liquid according to claim 1 or 2, which is characterized in that the antibacterials include blueness
Mycin, streptomysin and anphotericin；Wherein, the weight ratio of penicillin, streptomysin and amphotericin B is：0.2~0.6:0.1~
0.5:0.01~0.05.
8. adipose tissue Plantlet in vitro liquid according to claim 7, which is characterized in that the penicillin, streptomysin and two
The weight ratio of property mycin B is：0.5:0.3:0.02.
9. adipose tissue Plantlet in vitro liquid according to claim 1 or 2, which is characterized in that the phosphate buffer pH
Value is 7.2~7.6.
10. the preparation method of adipose tissue Plantlet in vitro liquid according to any one of claims 1 to 9, which is characterized in that including with
Lower step：
Aseptically, each component in claim 1 is mixed by formula, adipose tissue Plantlet in vitro liquid is prepared.
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| WO2023013596A1 (en) * | 2021-08-02 | 2023-02-09 | 株式会社Rainbow | Method for storing or transporting stem cells in unfrozen state |
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