CN104523753A - Preparation method, product and application of human umbilical cord mesenchymal stem cell cultural supernatant active factor and cell lysis buffer - Google Patents
Preparation method, product and application of human umbilical cord mesenchymal stem cell cultural supernatant active factor and cell lysis buffer Download PDFInfo
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Abstract
The invention discloses preparation and application of a human umbilical cord mesenchymal stem cell cultural supernatant active factor and cell lysis buffer. A conventional human umbilical cord mesenchymal stem cell culture medium is used for culturing a passage human umbilical cord mesenchymal stem cell, low-passage human umbilical cord mesenchymal stem cell cultural supernatant and cell lysis buffer are collected, and the collected supernatant and cell lysis buffer are prepared into lyophilized powder. Various cell factor mixtures with biological activity in the human umbilical cord mesenchymal stem cell cultural supernatant can be effectively preserved in the prepared lyophilized powder, and various cell factor mixtures are preserved in a mode of lyophilized powder; the bottleneck that the preservation period of the supernatant is limited can be effectively solved, and a foundation is established for industrialization of the human umbilical cord mesenchymal stem cell cultural supernatant.
Description
Technical field
The present invention relates to cytokine technical field, a specifically preparation method for human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid, the invention still further relates to the human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid and application thereof that are prepared by the method.
Background technology
The application of stem cell in beauty treatment is the utilization of stem cell in plastic surgery the earliest, and it is a kind of stem cell beautifying technique that research is more at present.And the stem cell kind applying more in plastic surgery is also mainly human umbilical cord mesenchymal stem cells, human adipose mesenchymal stem cells etc.
So-called stem cell cosmesis is traditionally all generally direct injection stem cell injection.And the source of this injection stem cell is mainly extracted from the embryo of miscarriage, so immunological rejection can be caused unavoidably, and there is certain reason problem.Even employing autologous stem cells, as human adipose mesenchymal stem cells, but also can introduce ectogenic albumen (hyclone) unavoidably in the cultivation amplification procedure of stem cell, easily cause immunological rejection.So scientists proposes a kind of new stem cell application process recently, the application of cells and supernatant active factors and cell pyrolysis liquid.
Research shows that cell culture supernatant contains and variously has bioactive cytokine, the immunological rejection of generation when its application can avoid stem cell to improve looks.But liquid culture supernatant is short in the room temperature holding time, and this just hinders its promotion and application.
In addition, for current stem cell sorting technology, the more common magnetic bead sorting being, but dividing of routine is chosen, magnetic bead together can carry out follow-up cultivation in company with stem cell, namely magnetic bead does not separate from stem cell, later stage like this for stem cell produces, inherently produce certain impact, although there is certain methods, as added some bacterium or other materials are degraded, be also equivalent to add the compositions such as other exogenous proteins like this, cause the secondary pollution to stem cell, think so actual not solve the problem.
Postscript, stem cell and culture thereof have many effects, but at present except application and insufficient, therefore, also need should be used as a popularization and development to it.
Summary of the invention
The object of the present invention is to provide the preparation method of a kind of human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid, assorting room is to cell not damaged, and incubation is without any animal sources composition, and the product high purity obtained, biological activity is high.
Another object of the present invention there are provided a kind of human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid, and purity is high, and biological activity is high, and storage life limit for length, and practicality is higher.
Another object of the present invention is to propose a kind of human umbilical cord mesenchymal stem cells culture supernatant active factors and the application of cell pyrolysis liquid in the product with biologic activity function, has high society and economy effect.
For achieving the above object, the invention provides following technical scheme:
A preparation method for human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid, comprises the following steps:
A) Healthy People umbilical cord mesenchymal stem cells is extracted in sorting;
B) through In vitro culture, amplification, a large amount of collection is passed at the low for the cells and supernatant of multiple and cell pyrolysis liquid;
C) lyophilized powder of cells and supernatant active factors and cell pyrolysis liquid is prepared by following peculiar step cryogenic vacuum.
Further, in step a, the sorting adopting magnetic lossless method for separating to carry out Healthy People umbilical cord mesenchymal stem cells is extracted, described magnetic lossless method for separating is: by wrapping up one deck sodium alginate on sorting magnetic bead, described sodium alginate is connected with can with the antibody of umbilical cord mesenchymal stem cells specific binding treating sorting, in conjunction with magnetite gathering after, more described sodium alginate to be degraded, namely obtains removing magnetic bead and sodium alginate umbilical cord mesenchymal stem cells.
Further, in step b, comprise following process: human umbilical cord mesenchymal stem cells is cultivated in 50ml culture bottle, when growing into 100% degree of converging, collect culture supernatant, and mix with isopyknic fresh culture, continue on for the amplification culture of cell, so repeatedly to passage to 10 generation, collect whole cell culture supernatant.
Further, in step b, umbilical cord mesenchymal stem cells goes down to posterity training number between 2-10.
Further, in step c, cryogenic vacuum preparation process specifically comprises: collect gained cells and supernatant and cell pyrolysis liquid respectively at 4 DEG C ,-20 DEG C ,-80 DEG C of gradient coolings are frozen solidify for subsequent use; Finally by-80 DEG C of frozen supernatant evacuation solidified, sublimation drying, removing ice crystal. finally obtained lyophilized powder ,-20 DEG C of preservations.
A kind of human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid that obtain is prepared according to described method.
Described human umbilical cord mesenchymal stem cells culture supernatant active factors and the application of cell pyrolysis liquid in the product with biologic activity function.
Compared with prior art, the invention has the beneficial effects as follows:
The beneficial effect of advantage of the present invention and generation is: human umbilical cord mesenchymal stem cells can secrete numerous protein in incubation, these protein can promote the growth of skin, regeneration and reparation, prove that it has definite biological activity in experiment in vitro, there is not the sale of such Related product in market. and the present invention collects the human mesenchymal stem cell culture supernatant and cell pyrolysis liquid of cultivating generation of passing at the low, and be prepared into lyophilized powder, effectively can solve supernatant storage life this bottleneck limited, for the commercial application of human mesenchymal stem cell culture supernatant is laid a good foundation, simultaneously, the present invention adopts magnetic lossless fast separating process to obtain primary human umbilical cord mesenchymal stem cells, reduce cell free time climbed out of from piece of tissue, incubation is not containing any animal sources composition, cryovacuum technology effectively can keep and use supernatant bioactie agent, such material is developed to the product with activity of cell biology function and (comprises malignant tumor at regenerative medicine, cardiovascular and cerebrovascular disease, immune disease and anti-ageingly to wait for a long time) and health product etc. in have broad application prospects, and by the great social benefit of generation and huge economic benefit.
Detailed description of the invention
Be clearly and completely described to the technical scheme in the embodiment of the present invention below, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Be described in more detail below in conjunction with the technical scheme of detailed description of the invention to this patent.
Embodiment
Technical scheme of the present invention is summarized as follows:
(1) separation and ientification of UCMSCs
A. the magnetic lossless of neonatal umbilical cord jelly of Wharton (Wharton ' s Jelly) mescenchymal stem cell is separated, process is: by wrapping up one deck sodium alginate on sorting magnetic bead, described sodium alginate is connected with can with the antibody of umbilical cord mesenchymal stem cells specific binding treating sorting, in conjunction with magnetite gathering after, after adding EDTA chelating agen, sodium alginate is degraded, and magnetic bead comes off, and collects and removes magnetic bead and sodium alginate umbilical cord mesenchymal stem cells, original cuiture and go down to posterity:
Under aseptic condition, it is several to get neonatal umbilical cord (length is no less than 20cm), puts umbilical cord preserving fluid, 4 DEG C of storages (time is no more than 8 hours).Rinse until intravenous is without bloodstain with D-Hanks liquid.Remove amniotic membrane epidermis and blood vessel, get jelly of Wharton (Wharton ' s Jelly) (also known as umbilical cord matrix), cut (cutting) broken piece of tissue for 1.5-2.5mm3 size, to be placed in the DMEM-LG cell culture fluid that 6 orifice plates contain (containing 10%FBS, bFGF 5ng/mL, L-glutaminate 2mM, penicillin 100U/mL, streptomycin 100 μ g/mL, and amphotericin B 1 μ g/mL), put 37 DEG C, cultivate in 5%CO2 incubator.Whole process strict aseptic technique.
Regenerative cell is adherent growth, after about 14 days when Growth of Cells merges to 90%-100%, changes fresh medium and removes non-adherent tissue particle.Then go down to posterity, trypsin with 0.25% is containing the EDTA of 1mmol/L) in 37 DEG C of digestion 3-5 minute, observe under inverted microscope, see cellular contraction, assemble, add the appropriate culture fluid containing 10% hyclone (FBS) immediately and stop digestion, then with suction pipe, cell is blown and beaten, dispersion, centrifugal, Trypan Blue exclusion test calculates living cells percentage rate, adjustment cell number with 1 × 106/ml density be inoculated in gelatin bag by after culture bottle, within 3 days, change liquid 1 time.When cell cover with into cause about 90%-100% merge time, can go down to posterity again according to upper method.After 3 generations, mesenchymal stem cell cryopreserving is for subsequent use in liquid nitrogen container.
(2) collection of culture supernatant:
Human mesenchymal stem cell is cultivated (10% autoserum+low sugar DMEM) in 10cm culture dish, when growing into 80-90% degree of converging, collect culture supernatant, and mix with isopyknic fresh culture, continue on for the amplification culture of cell, so repeatedly to passage to 10 generation, collect whole cell culture supernatant, for the preparation of follow-up lyophilized powder;
(3) preparation of lyophilized powder:
Collect the human mesenchymal stem cell culture supernatant of low passage number with 50ml sterile centrifugation tube in superclean bench, at 4 DEG C, the centrifugal 15min of 400g;
In superclean bench, open centrifuge tube, with in the collecting pipe that the supernatant in aseptic rifle head sucking-off centrifuge tube is aseptic, abandon precipitation, record mark ,-80 DEG C frozen solidify for subsequent use;
By-80 DEG C of frozen supernatant evacuation solidified, sublimation drying, removing ice crystal. finally obtained lyophilized powder ,-20 DEG C of preservations.
Namely human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid is prepared.
By this human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid system and other adjuvants or the combination such as reagent, health substance, obtain the product with biologic activity function.
Above the better embodiment of this patent is explained in detail, but this patent is not limited to above-mentioned embodiment, in the ken that one skilled in the relevant art possesses, various change can also be made under the prerequisite not departing from this patent aim.
Claims (7)
1. a preparation method for human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid, is characterized in that, comprises the following steps:
A) Healthy People umbilical cord mesenchymal stem cells is extracted in sorting;
B) through In vitro culture, amplification, a large amount of collection is passed at the low for the cells and supernatant of multiple and cell pyrolysis liquid;
C) lyophilized powder of cells and supernatant active factors and cell pyrolysis liquid is prepared by following peculiar step cryogenic vacuum.
2. the preparation method of human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid as described in the appended claim 1, it is characterized in that: in step a, the sorting adopting magnetic lossless method for separating to carry out Healthy People umbilical cord mesenchymal stem cells is extracted, described magnetic lossless method for separating is: by wrapping up one deck sodium alginate on sorting magnetic bead, described sodium alginate is connected with can with the antibody of umbilical cord mesenchymal stem cells specific binding treating sorting, in conjunction with magnetite gathering after, again described sodium alginate is degraded, namely obtain removing magnetic bead and sodium alginate umbilical cord mesenchymal stem cells.
3. the preparation method of human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid as described in the appended claim 1, it is characterized in that: in step b, comprise following process: human umbilical cord mesenchymal stem cells is cultivated in 50ml culture bottle, when growing into 100% degree of converging, collect culture supernatant, and mix with isopyknic fresh culture, continue on for the amplification culture of cell, so repeatedly to passage to 10 generation, collect whole cell culture supernatant.
4. the preparation method of human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid as described in the appended claim 1, is characterized in that: in step b, and umbilical cord mesenchymal stem cells goes down to posterity training number between 2-10.
5. the preparation method of human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid as described in the appended claim 1, it is characterized in that: in step c, cryogenic vacuum preparation process specifically comprises: collection gained cells and supernatant and cell pyrolysis liquid are respectively at 4 DEG C,-20 DEG C ,-80 DEG C of gradient coolings are frozen solidify for subsequent use; Finally by-80 DEG C of frozen supernatant evacuation solidified, sublimation drying, removing ice crystal. finally obtained lyophilized powder ,-20 DEG C of preservations.
6. human umbilical cord mesenchymal stem cells culture supernatant active factors and a cell pyrolysis liquid, is characterized in that: be prepared according to described method arbitrary in claim 1-5 and obtain.
7. human umbilical cord mesenchymal stem cells culture supernatant active factors and the application of cell pyrolysis liquid in the product with biologic activity function as recited in claim 6.
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| CN104946585A (en) * | 2015-07-07 | 2015-09-30 | 美天妮（天津）生物科技有限公司 | Production and freeze-drying method and application for supernate of culture solution of mesenchymal stem cell |
| CN105861432A (en) * | 2016-05-31 | 2016-08-17 | 安徽惠恩生物科技股份有限公司 | In vitro efficient amplification method for human umbilical cord blood hematopoietic stem cells |
| CN106109496A (en) * | 2016-07-06 | 2016-11-16 | 广东科玮生物技术股份有限公司 | Human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method |
| CN106244558A (en) * | 2016-08-31 | 2016-12-21 | 四川新生命干细胞科技股份有限公司 | The reprogramming of a kind of people's mononuclearcell is the method for induced multi-potent stem cell |
| CN106367389A (en) * | 2016-11-15 | 2017-02-01 | 东莞自然衡健康科技有限公司 | Preparation method and application of human umbilical cord mesenchymal stem cell factors |
| CN106727704A (en) * | 2016-12-26 | 2017-05-31 | 云南农业大学 | Dog stem cell secretion factor injection |
| CN106754639A (en) * | 2016-12-08 | 2017-05-31 | 北京银丰鼎诚生物工程技术有限公司 | A kind of mescenchymal stem cell factor large-scale producing method |
| CN109055308A (en) * | 2018-08-21 | 2018-12-21 | 启迪汉洱康（嘉兴）生物科技有限公司 | A kind of preparation method of polypeptide mixing stoste |
| CN117645972A (en) * | 2023-11-23 | 2024-03-05 | 新疆赛尔托马斯生物科技有限公司 | Preparation method of umbilical cord mesenchymal stem cell repair factor |
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| CN104946585A (en) * | 2015-07-07 | 2015-09-30 | 美天妮（天津）生物科技有限公司 | Production and freeze-drying method and application for supernate of culture solution of mesenchymal stem cell |
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| CN106109496B (en) * | 2016-07-06 | 2019-11-05 | 广东科玮生物技术股份有限公司 | Human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method |
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| CN106754639A (en) * | 2016-12-08 | 2017-05-31 | 北京银丰鼎诚生物工程技术有限公司 | A kind of mescenchymal stem cell factor large-scale producing method |
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| CN106727704A (en) * | 2016-12-26 | 2017-05-31 | 云南农业大学 | Dog stem cell secretion factor injection |
| CN109055308A (en) * | 2018-08-21 | 2018-12-21 | 启迪汉洱康（嘉兴）生物科技有限公司 | A kind of preparation method of polypeptide mixing stoste |
| CN117645972A (en) * | 2023-11-23 | 2024-03-05 | 新疆赛尔托马斯生物科技有限公司 | Preparation method of umbilical cord mesenchymal stem cell repair factor |
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Application publication date: 20150422 |