CN105797205A - Stem cell culture supernate gel and preparation method thereof - Google Patents
Stem cell culture supernate gel and preparation method thereof Download PDFInfo
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- CN105797205A CN105797205A CN201610248670.4A CN201610248670A CN105797205A CN 105797205 A CN105797205 A CN 105797205A CN 201610248670 A CN201610248670 A CN 201610248670A CN 105797205 A CN105797205 A CN 105797205A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0023—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0066—Medicaments; Biocides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/008—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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Abstract
The invention provides a stem cell culture supernate gel and a preparation method thereof.The gel is prepared from stem cell culture supernate and methylcellulose, and water servers as a solvent.The stem cell culture supernate is obtained by culturing mesenchymal stem cells with a stem cell culture medium containing no serum and then performing collecting and purifying.The preparation method of the gel includes the steps that firstly, the mesenchymal stem cells are cultured with the stem cell culture medium containing no serum, supernate is collected, and supernate I is obtained; the supernate I is centrifuged, ammonium sulfate is added into obtained supernate II, stirring, standing and centrifuging are performed, precipitate is taken, water is added for dissolving, and filtering is carried out; then, obtained protein filter liquor is purified with a gel column, and purified supernate is freeze-dried; finally, freeze-dried supernate is dissolved with sterile water, an obtained water solution is mixed with a methylcellulose water solution in an isopyknic mode, and the stem cell culture supernate gel is obtained.The stem cell culture supernate gel is used for repairing skin wounds and is significant in effect, and no cicatrix remains.
Description
Technical field
The invention belongs to medical biomaterial technical field, be specifically related to a kind of stem cell culture supernatant gel and preparation method thereof.
Background technology
The agglutination of wound surface is a complicated process, it relates to the process such as reinvent of the re-epithelialization at skin injury position, the hypertrophy of granulation tissue, inflammatory reaction, blood vessel hyperplasia and wound surface, any of process goes wrong, and all can cause wound healing obstacle.At present, the method for wound repairing is operation mainly, is about to open wound and is changed into Guan Bi wound by operation stitching, thus accelerating wound healing, but postoperative leave over cicatrix, there are the complication such as dysfunction after splitting again or healing in the appearance of wound overtension.Clinical position is frequently run onto some complex wound surface, such as the wound surface etc. after radioactivity wound surface, diabetic foot, life-time service hormone, these wound surface usually occur local blood circulation obstacle, fibroblastic biological activity reduce, the differentiation of mescenchymal stem cell and the negative effect such as paracrine action is suppressed, make wound surface promoting epidermization slowly, inflammatory reaction reduction, wound surface anti-infection ability decline, even if wound surface is less, adopt skin-grafting or the method Guan Bi wound surface directly sewed up, also Chang Buyi healing.In recent years; successively there are the launch such as the gel of cytokine, spray or dressing such as recombinant bovine or human fibroblastic growth factor (b-FGF), recombined human EGF, rhPDGF-BB and have been applied to clinic; shown by the clinical test results of multicenter large sample; the wound surface such as the burn wound of light degree Ⅱ～III degree, varicose ulcer of lower extremity, diabetic foot, radiation ulcer and decubital ulcer are adopted alone or in combination the therapeutic intervention of cytokine; wound healing can be accelerated, and the healing quality of wound surface can be significantly improved.But still suffer from many deficiencies: these somatomedin effects are single；After acting on wound surface, quickly degraded by the protein kinase in wound surface, it is impossible to effectively continuingly act on specificity target cell, thus causing that its bioavailability reduces, it is impossible to obtain satisfied clinical effectiveness.
Mescenchymal stem cell (MSC) is the one of adult stem cell, it to the reparation of cell with regeneration by tentative confirmation.Its pivotal role in wound healing includes in 2: one is the reconstruction directly participating in damage tissue, mainly include cell propagation, migrate, go back to the nest, differentiation etc.；Two is in the way of adjusting sexual function cell, indirectly participates in the reconstruction of tissue injury, relates generally to after stem cell is activated and secretes large number of biological bioactive molecule with paracrine form, promotes the function of other repair cells.For the former existing lot of experiments, the research for the latter also causes extensive concern recently.MSC can synthesize and secrete a large amount of cytokine and chemokines, such as substantial amounts of interleukin-6, interleukin 8, MCP-1, TIMP-1, GM-CSF, TGF-1, each collagen type (such as I, II, III, IV collagen type) and various lysozyme etc., these factors are extremely important in general wound.
Traditional cell expansion systems, mainly basal medium adds certain density hyclone, due to batch unstability and there is the risk polluting exogenous virus, therefore, adopt the serum-free medium containing serum substitute can avoid above-mentioned risk.
Summary of the invention
Solve the technical problem that: in order to overcome the problems such as tradition is high containing hyclone cell culture system risk, stem cell culture supernatant purification difficult, stem cell culture supernatant stability in use difference, the invention provides a kind of stem cell serum-free culture supernatant gel and preparation method thereof, this gel is used for skin wound reparation, and effect significantly and not stays cicatrix.
Technical scheme:
Stem cell culture supernatant gel, is made up of stem cell culture supernatant and methylcellulose, and solvent is water；Described stem cell culture supernatant is mescenchymal stem cell to use the stem cell media without serum collect purification after cultivating obtain.
The preparation method of described stem cell culture supernatant gel, comprises the following steps:
Step 1, uses the stem cell media without serum to cultivate mescenchymal stem cell, collects supernatant, obtain supernatant I；
Step 2, is centrifuged step 1 gained supernatant I, adds ammonium sulfate, stirring, standing, be centrifuged, take precipitation, be dissolved in water, filter, obtain albumen filtrate in gained supernatant II；
Step 3, by step 2 gained albumen filtrate through gel column purification, obtains purification supernatant；
Step 4, by step 3 gained purification supernatant lyophilizing, obtains lyophilizing supernatant；
Step 5, dissolves step 4 obtained freeze-drying supernatant sterilized water, and obtained aqueous solution mixes with methylated cellulose aqueous solution equal-volume, to obtain final product.
Further, in step 2 centrifugal condition be followed successively by 7000rpm-8000rpm, 4 DEG C, 10min-20min and 9000rpm-10000rpm, 4 DEG C, 10min-20min.
Further, step 2 adds ammonium sulfate step to carry out in 0 DEG C of ice bath.
Further, in step 2, static conditions is 4 DEG C, 12h-16h.
Further, the gel column adopted in step 3 is glucosan G-25 gel column, and eluant is 0.1M-0.15MNaCl aqueous solution.
Beneficial effect:
The stem cell media supernatant gel of the present invention is used for skin wound reparation, and effect is notable, and does not stay cicatrix.
Accompanying drawing explanation
Fig. 1 is the dyeing in 15 days of test example small mouse union of wounded skin；
Fig. 2 is the Masson dyeing in 15 days of test example small mouse union of wounded skin；
Wherein, in figure, arrow indication is wound surface position.
Detailed description of the invention
Embodiment 1
The preparation of mescenchymal stem cell: take fresh and healthy neonatal umbilical cord, rinsed well by umbilical cord with PBS, remove blood vessel, strips out the Fahrenheit glue tissue of the inside, fully shreds gained tissue to 1mm3Size, adds serum-free medium, in 37 DEG C, 5%CO2Incubator is cultivated, after cell covers with, goes down to posterity with 0.25% trypsinization.Take the 3rd generation umbilical cord derived mesenchymal stem cell standby, to prepare gel.
Serum-free medium main formula: human serum albumin: 4g/L-10g/L, glycine: 30.00mg/L, non essential amino acid: 1mg/L-50mg/L, recombinant human insulin: 1mg/L-10mg/L, human transferrin: 5mg/L-20mg/L.
The preparation method of stem cell serum-free culture supernatant gel, comprises the following steps:
Step 1, takes the 3rd generation umbilical cord derived mesenchymal stem cell serum-free medium and cultivates, when covering with 70% in culture bottle, be further cultured for 48 hours, collect supernatant；
nullStep 2，Take 500mL step 1 gained supernatant，8000rpm、4 DEG C of centrifugal 15min，Remove piece of tissue therein and other impurity，It is placed in the beaker of 1L，The 304g ammonium sulfate solids being slowly added to weigh in advance is (in 85% saturation，With reference to ammonium sulfate precipitation agent table)，Limit edged magnetic stirrer，At least 1h adds，More than operate and complete in 0 DEG C of frozen water mixing bath，4 DEG C of chromatography cabinets continue stirring to being completely dissolved，Chromatography cabinet stands 12h，9000rpm、4 DEG C of centrifugal 10min collect albumen precipitation，It is dissolved in water and is settled to 400mL，With filter (adding 2 0.45 μm of filter membranes) filtrate protein solution，Again with sterilizing deionized water rinsing filter membrane several times to reduce the loss of albumen，Final albumen filtrate volume is about 540mL；
Step 3, glucosan G-25 gel column (5 × 60cm) is connected AKTA protein purification instrument, with the deionized water containing 0.15MNaCl as eluant, regulate flow velocity 10mL/min, each sample introduction 80mL, until ultraviolet absorption value substantially rises, start to connect sample until no longer there being uv absorption, the sample collected is preserved at 4 DEG C with after 0.22 μm of membrane filtration, obtaining purification supernatant, measure through BCA method, the protein concentration in gained purification supernatant is 1.3mg/mL, it is that 3% methylated cellulose aqueous solution equal-volume mixes with concentration, obtains gel (low dosage)；
Step 4, by step 3 gained purification supernatant lyophilizing, lyophilizing program such as table 1, obtains lyophilizing supernatant；
Step 5, dissolves step 4 obtained freeze-drying supernatant sterilized water, and in lyophilizing supernatant water solution, protein concentration is 19mg/mL, is that 3% methylated cellulose aqueous solution equal-volume mixes with concentration, obtains gel (high dose).
Table 1 purification supernatant lyophilizing program
Test example ICR mouse species skin injury healing assay
1.1 experiment reagents and material
Experiment reagent: lyophilizing supernatant, methylcellulose, picric acid, 4% chloral hydrate, povidone iodine.
Experiment material: skin test needle, swab stick, shaver, sterile gauze, 5mm skin puncher, operating theater instruments (straight peen operating scissors, tweezers), mouse cage, healthy SPF Grade I CR mice 40, balance, beaker, mask, glove.
The preparation of main agents
3% methylcellulose: put into 80-90 DEG C of hot water of requirement in container, is gradually added into methylcellulose under slowly stirring, is placed in and allows it dissolve gel then on ice；
Gel (low dosage): purify supernatant equal-volume with 1.3mg/mL with 3% methylcellulose and mix；
Gel (high dose): mix with 19mg/mL lyophilizing supernatant equal-volume with 3% methylcellulose.
1.2 experimental techniques
Packet: laboratory animal is randomly divided into blank group (10), solvent control group (10) and the big group of experiment material group (20) three, and wherein experiment material group is divided into again 2 groups, and the sample number often organized is 10.
1. blank group: 10, skin of back damages, and during experiment, wound does not give any process；
2. solvent control group: 10, skin of back damages, and during experiment, wound gives 3% methylcellulose process；
3. experiment material group: totally 20, is divided into I group, II group
I group: 10, skin of back damages, and during experiment, wound gives gel (low dosage) process.
II group: 10, skin of back damages, and during experiment, wound gives gel (high dose) process.
The foundation of skin injury animal model: anesthesia ICR mice, prostrate it is fixed on operating-table, back is removed by hair with shaver, 5mm skin puncher is adopted to prepare skin trauma model in mouse back, require damage deep and fascia layer, wound surface is the circle of diameter 0.5cm, and be administered once every day (gel, 50 μ L/ time) at regular time and quantity.
Often group after wound 1,3,5,7,9d take 5 ICR mices at random and carry out wound surface and take pictures, use ImageJ image analysis software, calculate the size of healing wound surface.
Wound healing rate=(size of the initial wound surface size-wound surface that do not heal)/initial wound surface size × 100%
Result is as follows:
The healing rate of wound surface is as shown in table 2 in different time points, the three of different time points groups of Wound healing rates are carried out paired t-test with blank group respectively, the 5th of wound healing, 7 days, high dose group Wound healing rate is more than matched group, its difference statistically significant (P < 0.05), 3rd, 9 days, Wound healing rate was without significant difference.
Table 2 different time points ICR wounds in mice healing rate (%)
Histologic analysis
1.HE dyes
The each group of incision 1cm scope ring layer skin histology containing both sides of leaving and taking taking the 3rd, 6,8,15 days carries out paraffin section and makes and carry out HE dyeing, assesses the general condition of different group wound healing.
As it is shown in figure 1, visible through conventional HE dyeing, 3d blood crusts separates with the edge of wound skin thickened, and wound surface is significantly granulation tissue formation as seen, and fusiformis fibroblast starts hypertrophy, and each group difference is inconspicuous.6d experimental group fibroblast quantity is many compared with matched group, and on the whole, cell quantity reduces.Each group mice wound still has thick crust, but has started to peel off wound.Obvious epithelization around 8d wound surface, wound surface area is obviously reduced, experimental group (high dose and low dose group), has been filled with whole wound area, and the visible granulation tissue of matched group is formed, but imperfect.15d wound heals substantially, and original wound is covered by epidermis completely, and epidermis homogenizing is thinning.Low dose group wound width is significantly less than matched group, and high dose group wound cell level becomes many, and closer to normal skin tissue, the skin accessory organs such as hair follicle sweat gland occurs in minority high dose group individuality wound.
2.Masson dyes
The each group of incision 1cm scope ring layer skin histology containing both sides of leaving and taking taking the 3rd, 6,8,15 days carries out paraffin section and makes and carry out Masson dyeing, assesses the general condition that in different group wound healing process, collagen is formed.
As in figure 2 it is shown, the wound surface cambium section gloomy trichrome stain of horse shows (n=3), after Masson dyeing wound, 3d-8d, granulation tissue occurs, collagen fiber hyperplasia is inconspicuous.15d after wound, collagen fiber hyperplasia is notable.Blank group and the postoperative 15d of MC group, neonatal dermal collagen accumulation is loose, and many in filling the air shape, the collagen protein of rule accumulation is less.Low dosage and high dose group corium have collagen fiber that are thick and that substantially move towards, and dermal collagen accumulation is better than matched group.
Claims (6)
1. stem cell culture supernatant gel, it is characterised in that: being made up of stem cell culture supernatant and methylcellulose, solvent is water；Described stem cell culture supernatant is mescenchymal stem cell to use the stem cell media without serum collect purification after cultivating obtain.
2. the preparation method of the stem cell culture supernatant gel described in claim 1, it is characterised in that: comprise the following steps:
Step 1, uses the stem cell media without serum to cultivate mescenchymal stem cell, collects supernatant, obtain supernatant I；
Step 2, is centrifuged step 1 gained supernatant I, adds ammonium sulfate, stirring, standing, be centrifuged, take precipitation, be dissolved in water, filter, obtain albumen filtrate in gained supernatant II；
Step 3, by step 2 gained albumen filtrate through gel column purification, obtains purification supernatant；
Step 4, by step 3 gained purification supernatant lyophilizing, obtains lyophilizing supernatant；
Step 5, dissolves step 4 obtained freeze-drying supernatant sterilized water, and obtained aqueous solution mixes with methylated cellulose aqueous solution equal-volume, to obtain final product.
3. the preparation method of stem cell culture supernatant gel according to claim 2, it is characterised in that: in step 2 centrifugal condition be followed successively by 7000rpm-8000rpm, 4 DEG C, 10min-20min and 9000rpm-10000rpm, 4 DEG C, 10min-20min.
4. the preparation method of stem cell culture supernatant gel according to claim 2, it is characterised in that: step 2 adds ammonium sulfate step and carries out in 0 DEG C of ice bath.
5. the preparation method of stem cell culture supernatant gel according to claim 2, it is characterised in that: in step 2, static conditions is 4 DEG C, 12h-16h.
6. the preparation method of stem cell culture supernatant gel according to claim 2, it is characterised in that: the gel column adopted in step 3 is glucosan G-25 gel column, and eluant is 0.1M-0.15MNaCl aqueous solution.
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Cited By (7)
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| CN106938054A (en) * | 2017-04-21 | 2017-07-11 | 芜湖扬展新材料科技服务有限公司 | A kind of preparation method of placenta stem-cell composite bioactivity glass dressing |
| CN107308441A (en) * | 2017-06-15 | 2017-11-03 | 江苏迈健生物科技发展股份有限公司 | Stem cell culture supernatant gel combination and its preparation method and application |
| CN107468708A (en) * | 2017-08-10 | 2017-12-15 | 山东景源生物科技有限公司 | A kind of preparation method of Stem Cell Activity factor gel and the application in Hard agglut wound treatment |
| CN108517002A (en) * | 2018-04-20 | 2018-09-11 | 命之本源医疗科技（北京）有限公司 | A kind of stabilizer of stem cell culture supernatant secretory protein |
| CN111117948A (en) * | 2020-01-15 | 2020-05-08 | 安徽瑞达健康产业有限公司 | Fibroblast culture method |
| CN113491713A (en) * | 2021-06-24 | 2021-10-12 | 四川大学华西医院 | Adipose-derived mesenchymal stem cell culture supernatant gel and preparation method thereof |
| CN114540290A (en) * | 2022-02-21 | 2022-05-27 | 赛尔生命科技(深圳)有限公司 | Supernatant gel stem cell culture medium for repairing bone injury and preparation method thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106938054A (en) * | 2017-04-21 | 2017-07-11 | 芜湖扬展新材料科技服务有限公司 | A kind of preparation method of placenta stem-cell composite bioactivity glass dressing |
| CN107308441A (en) * | 2017-06-15 | 2017-11-03 | 江苏迈健生物科技发展股份有限公司 | Stem cell culture supernatant gel combination and its preparation method and application |
| CN107468708A (en) * | 2017-08-10 | 2017-12-15 | 山东景源生物科技有限公司 | A kind of preparation method of Stem Cell Activity factor gel and the application in Hard agglut wound treatment |
| CN108517002A (en) * | 2018-04-20 | 2018-09-11 | 命之本源医疗科技（北京）有限公司 | A kind of stabilizer of stem cell culture supernatant secretory protein |
| CN111117948A (en) * | 2020-01-15 | 2020-05-08 | 安徽瑞达健康产业有限公司 | Fibroblast culture method |
| CN113491713A (en) * | 2021-06-24 | 2021-10-12 | 四川大学华西医院 | Adipose-derived mesenchymal stem cell culture supernatant gel and preparation method thereof |
| CN114540290A (en) * | 2022-02-21 | 2022-05-27 | 赛尔生命科技(深圳)有限公司 | Supernatant gel stem cell culture medium for repairing bone injury and preparation method thereof |
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| CN105797205B (en) | 2018-08-31 |
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