CN108617639A

CN108617639A - A kind of cells frozen storing liquid of human adipose-derived stem cell

Info

Publication number: CN108617639A
Application number: CN201810470135.2A
Authority: CN
Inventors: 李倩
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-03-06
Filing date: 2016-03-06
Publication date: 2018-10-09
Also published as: CN108633876A; CN108450459A; CN107232186A; CN108450460A; CN107232186B; CN108541701A; CN105494317B; CN105494317A; CN109090097A; CN108633875A; CN108541700A

Abstract

The present invention provides a kind of human adipose-derived stem cell frozen stock solutions and preparation method thereof.The cells frozen storing liquid includes low-density lipoprotein, trehalose, glycerine, lecithin, polypeptide, bFGF, vitamin E, reductive glutathione.Cells frozen storing liquid provided by the invention in particular improves the survival rate after freeze-stored cell recovery for freezing human adipose-derived stem cell.With preferable application prospect.

Description

A kind of cells frozen storing liquid of human adipose-derived stem cell

The present invention is that application No. is " 201710643072.1 ", a kind of entitled " cells of human adipose-derived stem cell The divisional application of frozen stock solution " patent of invention.

Technical field

The present invention relates to histocyte culture technique fields, and in particular to a kind of human adipose-derived stem cell frozen stock solution and its preparation Methods and applications.

Background technology

Fat stem cell (Adipose-derived stem cells, ADSCs) is to be detached from adipose tissue in recent years A kind of obtained stem cell with multi-lineage potential, has the characteristics that the general stem cell such as to expand, be not easy aging rapidly. Human adipose-derived stem cell, that is, body fat mescenchymal stem cell is be now widely used for organizational project and regenerative medicine field one Kind adult stem cell has multi-lineage potential as mesenchymal stem cell.ADSCs is grown in fibroblast sample, born of the same parents Slurry and kernel are abundant, are arranged in parallel or whirlpool sample.Cell cycle analysis shows that the cell of G0/G1 phases accounts for 69%, the S phases and accounts for 24%, the G2/M phase account for 8%.2-3 days I times of the cell Proliferations of secondary culture under the existence condition of fetal calf serum.Repeatedly passage (10- 20 generations) after, there is senile cell, the 15th generation cell mass after passing on 6 times without obviously slowing down in cell colony in cell proliferation rate Senile cell accounts for about 15% in body.

Since human adipose mesenchymal stem cells are separately cultured, the period is longer, about 2 weeks time that primary cell is paved with or more, reaches 3 weeks or so are needed to certain quantity.The passage of human adipose mesenchymal stem cells longterm culture in vitro is easy to happen Spontaneous Differentiation, loses Remove the potential of its Multidirectional Differentiation.So to ensure the continuity of experiment, it is necessary to provide a large amount of experiment or used in tissue engineering at any time Seed cell.Therefore, strongly for the demand of cell.Now, the cell produced is subjected to freezen protective, when use It is several again to thaw using the use pattern for being also routine.However the frozen stock solution that stem cell cryopreserving uses in the prior art has containing serum With two classes of serum-free, since serum has the shortcomings that complicated component, unstable quality, expensive, serum-free freezes and answers Soviet Union's technology becomes development trend.Cord blood stem cell relies primarily on the protection of anti frozen liquid dimethyl sulfoxide (DMSO) (DMSO) and serum. Commonly used cell-protecting also has hydroxyethyl starch 0ES), polyethylene glycol (PEG) etc..But DMSO can generate poison to cell Property effect, irreversible damage is caused to the stem cell frozen.To obtain the neural stem cell of convenient sources, carry out effective god The urgent demand in this field through stem cell cryopreserving method, establish a kind of prolonged cold preserve the method for human adipose-derived stem cell for Grinding for human adipose-derived stem cell is promoted to make internal disorder or usurp and using being of great significance.

Invention content

The technical problem to be solved in the present invention is to provide a kind of human adipose-derived stem cell freezen protective liquid and corresponding preparations Method and application method.

Red fruit Huang meat nanmu is a kind of dungarunga, and applicant passes through the study found that having the object for capableing of cold resistant in the plant Matter exists, and therefore, applicant is obtained by high flux screening has cold-resistant polypeptide accordingly, and therefore, applicant attempts will It is added in freezen protective liquid, for preserving cell.

Technical scheme is as follows：

A kind of human adipose-derived stem cell freezen protective liquid, it is characterised in that be grouped as by following each group：It is characterized in that freezing is deposited Contain in liquid:0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1%w/ V, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, the sequence such as SEQ ID NO of the polypeptide：Shown in 1-32 is any.The polypeptide has applicant a large amount of by early period Library immunoscreening obtains, and the polypeptide has the function of protecting cells from damage.Its title be respectively KATS-1, KATS-2, KATS-3、KATS-4、KATS-5、KATS-6、KATS-7、KATS-8、KATS-9、KATS-10、KATS-11、KATS-12、 KATS-13、KATS-14、KATS-15、KATS-16、KATS-17、KATS-18、KATS-19、KATS-20、KATS-21、KATS- 22、KATS-23、KATS-24、KATS-25、KATS-26、KATS-27、KATS-28、KATS-29、KATS-30、KATS-31、 KATS-32 is corresponding in turn in SEQ ID NO：1-32.

In the frozen stock solution of the cell of the present invention, trehalose and vitamin E and polypeptide, which have, explicitly resists external wound Damage of the evil to cell, especially polypeptide also have and protect cells from damage of the ice crystal to cell when freezing.

The present invention also provides a kind of preparation methods of the frozen stock solution of human adipose-derived stem cell, i.e., mix each component and shake Even.

The present invention also provides a kind of human adipose-derived stem cell cryopreservation methods, include the following steps:

A. prepared by frozen stock solution:The cells frozen storing liquid is prepared, by each component according to conventional operating method by each component It can be obtained according to the mixing of corresponding ratio；

B. prepared by cell suspension:Culture grows into one bottle of people's adipocyte of single layer, and 0.20% trypsin solution digests 4 points Clock abandons trypsin solution, and DMEM culture solution 15ml are added, and gently being blown and beaten with suction pipe keeps cell uniform, centrifuges 4000 revs/min, abandons supernatant, Step A frozen stock solution 2ml are added, is mixed, is placed in sterile cryopreservation tube；

C. it freezes:Cryopreservation tube is first frozen into 30min at 4 DEG C, -30 DEG C is then placed in and freezes lh, -80 DEG C is placed into and freezes 3h is finally moved into liquid nitrogen and is preserved；

D. cell recovery:It takes out cryopreservation tube and is quickly placed into 37 DEG C of water-baths, concussion is melted up to cell suspension, then used completely 5 times of DMEM liquid dilutions, 1000r/min centrifuge 5min, and centrifugation removal supernatant repeats 3 times.The cell suspended concentration is 10⁸Cell/ml-10¹⁰Cell/ml.

Beneficial effects of the present invention:

The human adipose-derived stem cell frozen stock solution of the present invention, recovery cell survival rate relatively use regular growth up to 97% or more The recovery survival rate of frozen stock solution is obviously improved, and there is no the loss of cell.

The stem cell cryopreserving liquid of the present invention can not be changed with long-term preservation stem cell, cell activity, ensure that cell Biological activity.

Nerve cell cryopreservation methods of the present invention, operation is simple and feasible, affordable, has preferable practical value.

Specific implementation mode

The preparation of 1 polypeptide of embodiment

Red fruit Huang meat nanmu blade 5g is taken, is cleaned up, rubs, squeezes the juice, papain and trypsase, enzyme concentration is added 8000IU/g blades, 47 DEG C of hydrolysis temperature, pH value 7.5, enzymolysis time 1.5h, 90 DEG C of enzyme deactivation 10min after the completion of enzymolysis；After enzyme deactivation Material filtering remove insoluble matter, obtain solution, 4% activated carbon adsorption decoloration is added in gained polypeptide solution, with glucan G-50 (Sephadex G-50) carries out peptide separation, the elution of 20mmol/L HCl solutions, and flow velocity 1.3mL/ minutes is collected different respectively The eluted product of period adjusts solution to pH7.0, and 10000 revs/min centrifuge 15 minutes, through macroreticular resin DA201-C desalinations It after processing, is concentrated in vacuo, supernatant freeze-drying is spare；Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), the band of small-molecular-weight is recycled, wherein passing through functional verification, the sequence that 32 small peptides are obtained is fatty with people is stablized The effect of stem cell promotes stem cell growth, keeps stem cell normal growth state.According to peak separation different in chromatographic column Time can obtain corresponding small peptide in batches, also can the artificial synthesized polypeptide.The sequence of the polypeptide such as SEQ ID NO： Shown in 1-32.It is respectively designated as KATS-1~32.

The preparation of 2 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 1.

The preparation of 3 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 2.

The preparation of 4 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 3.

The preparation of 5 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 4.

The preparation of 6 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 5.

The preparation of 7 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 6.

The preparation of 8 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 7.

The preparation of 9 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 8.

The preparation of 10 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 9.

The preparation of 11 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 10.

The preparation of 12 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 11.

The preparation of 13 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 12.

The preparation of 14 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 13.

The preparation of 15 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 14.

The preparation of 16 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 15.

The preparation of 17 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 16.

The preparation of 18 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 17.

The preparation of 19 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 18.

The preparation of 20 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 19.

The preparation of 21 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 20.

The preparation of 22 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 21.

The preparation of 23 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 22.

The preparation of 24 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 23.

The preparation of 25 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 24.

The preparation of 26 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 25.

The preparation of 27 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 26.

The preparation of 28 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 27.

The preparation of 29 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 28.

The preparation of 30 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 29.

The preparation of 31 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 30.

The preparation of 32 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 31.

The preparation of 33 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1% W/v, bFGF 1%w/v, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to DMEM culture mediums 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 32.

The preparation of 34 human adipose-derived stem cell frozen stock solution of embodiment

By 0.5%w/v low-density lipoproteins, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, bFGF1%w/ V, vitamin E 1%w/v, reductive glutathione 0.5%w/v are finally settled to 100ml with DMEM culture mediums.

35 comparative example of embodiment

With the serum-free frozen stock solution that is authorized in CN 102550542B frozen stock solution as a contrast.

The compliance test result of 36 frozen stock solution of embodiment

Cells frozen storing liquid prepared by above example 2-34 and comparative example, carries out cell cryopreservation by the following method respectively And recovering experiment.

Cell cryopreservation process:

Culture grows into the human adipose-derived stem cell of single layer, cell density about 6*10⁹A/ml, the PBS that pH7.0 is added are washed Cell surface is primary.

0.20% trypsin solution of cell is digested 4 minutes, trypsin solution is abandoned, DMEM culture solution 15ml are added, use suction pipe Gently piping and druming keeps cell uniform, centrifuges 4000 revs/min, abandons supernatant, and the frozen stock solution prepared by embodiment 1-33 and comparative example 1 is added 2ml is mixed, and is placed in sterile cryopreservation tube；

Freezing:Cryopreservation tube is first frozen into 30min at 4 DEG C, -30 DEG C is then placed in and freezes lh, -80 DEG C is placed into and freezes 3h is finally moved into liquid nitrogen and is preserved；It freezes respectively 1 week, 4 months, 8 months.Every group of 3 repetitions.

Cell recovery:It takes out cryopreservation tube and is quickly placed into 37 DEG C of water-baths, concussion is until cell suspension melts completely, then with 5 The dilution of times DMEM liquid, 1000r/min centrifuge 5min, and centrifugation removal supernatant repeats 3 times, calculates cell survival rate.As a result such as Under：

As can be seen from the above results, frozen stock solution of the invention, particularly suitable for the culture that freezes of human adipose-derived stem cell, tool There is preferable cell protection activity.

37 stem cell antigen of embodiment detects

Fat stem cell has a variety of specific antigens and receptor, mainly have 3,13, D29,34,45, CD49e, CD59, CD73, CD90, CD105, HLA-ABC etc..

Example 36 freezes 4 months fat stem cells, removes culture solution, with 1:1 2.5% trypsin solution With 0.0296EDTA solution mixture slakings, every 100ul is made after being washed with the PBS containing 1% bovine serum albumin(BSA) (BSA) and contains 1* 1O6 single cell suspension is divided into 7 parts, be separately added into 7 Eppendorf pipes and number, and totally 20 μ L are added in No.1 pipe FITC Mouse IgGl, APC_CY7Mouse IgG2b and dye solution are for detecting since antibody non-specific binding is produced As a contrast, other test tubes are separately added into CD29, CD34,44,45,105, each 20 μ L of HLA.DR monoclonal antibodies to raw background, Often pipe is separately added into 100 μ L of cell suspension (containing 1*106 cell), is incubated at room temperature 25min, after being washed with the PBS containing 1%BSA, Flow cytomery.Analysis result:Six kinds of surface antigens 29,34,44, CD45 of flow cytometry analysis human adipose-derived stem cell, CD105 and HLA.DR, the result shows that there is human adipose-derived stem cell characteristic using the cell that embodiment 2-34 medium cultures go out. HLA.DR is negative, and it is fibroblast to exclude such cell.

38 adipogenic induction of embodiment and detection

Since fat stem cell has Multidirectional Differentiation ability, differentiation is carried out to fat stem cell under certain conditions and is lured It leads, the differentiated cell of specific function can be obtained.

Example 36 freezes 4 months fat stem cells, and dexamethasone is added into culture solution, uses general method Adipogenic induction is carried out to fat stem cell.By cultivate find, all stem cells frozen can to Adipocyte Differentiation, And it is substantially all to be all divided into adipocyte, there is higher activity.This absolutely proves that the fat stem cell after freezing is still So retain original cell activity.

Above-described is only some embodiments of the present invention.For those of ordinary skill in the art, not Under the premise of the disengaging present invention makes design, several changes and improvements can also be made, these belong to the protection domain of invention.

Sequence table

<110>Li Qian

<120>A kind of cells frozen storing liquid of human adipose-derived stem cell

<160> 32

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 1

Leu Met Ser His Cys Gln Met His Glu Pro His Cys Pro Ala Ile Gly

1 5 10 15

Val Trp

<210> 2

<211> 18

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 2

Ile Ser Arg Trp Glu Thr Arg Ile Glu Asn Gly Val Phe His Asn Pro

1 5 10 15

Gly Tyr

<210> 3

<211> 18

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 3

Tyr Cys Glu Gln Gln Asn His Arg Thr Cys Asp Ala Val Gly Lys Leu

1 5 10 15

Ile Gln

<210> 4

<211> 18

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 4

Gly Trp Met Met Cys Ile Asp Pro Ile Met Pro Gly Met Gln Ala Met

1 5 10 15

Gln Lys

<210> 5

<211> 18

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 5

Pro Arg Arg Ser Met Arg Tyr Arg Arg Leu Ala Trp Ser Gln Ser Leu

1 5 10 15

Pro Phe

<210> 6

<211> 18

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 6

Gln Asp Ser Arg Ile Phe Arg Lys Trp Ile Gln Gly Gln Ser Tyr Tyr

1 5 10 15

Gln Phe

<210> 7

<211> 18

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 7

Gly Asp Ile Leu Arg Pro Lys Asn Phe Tyr Arg Trp Ser Lys Ala Asp

1 5 10 15

Val Gly

<210> 8

<211> 18

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 8

Phe Gln His Cys Asn Tyr Gln Tyr Glu Met Ser Leu Trp Ala Glu Gln

1 5 10 15

Cys Tyr

<210> 9

<211> 17

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 9

Arg Lys Thr Thr Lys Arg Lys Cys Lys Gly Gly Glu Gln Gly Arg Gln

1 5 10 15

Ala

<210> 10

<211> 16

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 10

Val Leu Tyr Asp Asn Arg Pro Trp Gln Ser Ala Arg Gln Ser Glu Arg

1 5 10 15

<210> 11

<211> 17

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 11

Asp Ile Ser Tyr Gly Asn Arg Arg Ser Gly Gly Phe Lys Ser Glu Gly

1 5 10 15

Ala

<210> 12

<211> 16

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 12

Cys Asp Ile Gly Arg Gly Pro Ala Ser Cys Asp Ile Thr Val Gln Ile

1 5 10 15

<210> 13

<211> 17

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 13

Pro Trp Ser Ile Thr Pro Ile Met Cys Arg Arg Gly Arg Arg Ile Phe

1 5 10 15

Ser

<210> 14

<211> 16

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 14

Arg His Trp Ala Met Pro Asn Gln Cys Arg Gln Cys Tyr His Asn Phe

1 5 10 15

<210> 15

<211> 18

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 15

Ala Met Gln Asp Lys Ala Glu Ser Phe Leu Leu Asp Leu Lys Ser Ser

1 5 10 15

Glu Trp

<210> 16

<211> 17

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 16

Ile Arg Thr Trp Arg Leu Cys Gly Cys Asn Ile Arg His Met Phe Ile

1 5 10 15

Gly

<210> 17

<211> 16

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 17

Arg Val Thr Gln Asn Ile Trp Asn Trp Gln Leu Thr Ile Leu Gln Cys

1 5 10 15

<210> 18

<211> 18

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 18

Glu Met Phe Lys Val Lys Asn Asp Val Thr Trp Tyr Ser Tyr Gln Trp

1 5 10 15

Gly Ser

<210> 19

<211> 17

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 19

Gln Phe Gln His Leu Gly Trp Gln Val Asp Arg Asn Met Arg Glu Lys

1 5 10 15

Asp

<210> 20

<211> 16

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 20

Arg Met Asn Ala Gln Leu Glu Asp Lys Leu Thr Phe Leu Met Ile Glu

1 5 10 15

<210> 21

<211> 17

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 21

Lys Gly Ser Arg Pro Trp Arg Pro Phe Gln Gly Pro Met Arg Asp Ile

1 5 10 15

Asp

<210> 22

<211> 16

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 22

Glu Gln Glu Pro Leu His Pro Pro Gln His Gly His Gln Ser Thr Ser

1 5 10 15

<210> 23

<211> 18

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 23

Lys Cys Asn Pro Gln Ser Met Val His Thr Glu Asp Trp Gln Pro Tyr

1 5 10 15

Tyr Trp

<210> 24

<211> 17

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 24

Ala Ala Ser Thr Gly His Ala Met His Tyr Gln Glu Phe Phe Asn Gly

1 5 10 15

Ala

<210> 25

<211> 16

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 25

Asn Asp Ser Asp Trp Ile Ser Gly Ser Phe Asp Glu Gln Asn His Gln

1 5 10 15

<210> 26

<211> 17

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 26

Arg Gly Ser Ile Ala Gly Gln Gly Arg Asp Thr Trp Asp Met Leu Gly

1 5 10 15

Pro

<210> 27

<211> 16

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 27

Cys Cys Gln Trp Tyr Thr Arg Thr Val Gln Arg Met Arg Pro Arg Thr

1 5 10 15

<210> 28

<211> 17

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 28

Gln Gln Glu His Ser Gln Pro Ser His Leu Gln Ser Thr Ile Gly Cys

1 5 10 15

Arg

<210> 29

<211> 16

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 29

Thr Ala Leu Ser Arg Phe Trp His Met Glu Pro Ser His Arg Arg Phe

1 5 10 15

<210> 30

<211> 17

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 30

Ser Glu Thr Lys Gly Gln Phe Arg Thr Gly Arg His Thr Gln Ala Gly

1 5 10 15

Gln

<210> 31

<211> 16

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 31

Pro Ser Arg Gly Tyr Pro Cys Asp Trp Gly Asp Val Gln Pro Pro Trp

1 5 10 15

<210> 32

<211> 17

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 32

Gly Val Thr Phe Gln Cys Met Asn Asn Gly Ile Ile Gln Thr Tyr Glu

1 5 10 15

Gln

Claims

1. a kind of human adipose-derived stem cell frozen stock solution, which is characterized in that it is characterized in that being grouped as by following each group：0.5%w/v is low Density lipoprotein, the trehalose of 1%w/v, 1% glycerine, 1%w/v lecithin, polypeptide 0.1%w/v, bFGF 1%w/v, dimension life Plain E 1%w/v, reductive glutathione 0.5%w/v are finally settled to 100ml with DMEM culture mediums.

2. the preparation method of human adipose-derived stem cell frozen stock solution described in claim 1, which is characterized in that including mixing the formula The component of ratio obtains the frozen stock solution.

3. application of the human adipose-derived stem cell frozen stock solution described in claim 1 after improving freeze-stored cell recovery in survival rate.

4. human adipose-derived stem cell frozen stock solution as described in claim 1, it is characterised in that：The polypeptide sequence such as SEQ ID NO: Shown in 7.

5. a kind of polypeptide, it is characterised in that：Sequence such as SEQ ID NO:Shown in 7.