CN108450459A - A kind of cells frozen storing liquid of human adipose-derived stem cell - Google Patents

Abstract

The invention provides a human adipose stem cell cryopreservation solution and a preparation method thereof. The cell cryopreservation solution includes low-density lipoprotein, trehalose, glycerin, lecithin, polypeptide, bFGF, vitamin E, and reduced glutathione. The cell cryopreservation solution provided by the invention is used for cryopreserving human adipose stem cells, and especially improves the survival rate of the cryopreserved cells after recovery. It has a good application prospect.

Description

A cell cryopreservation solution for human fat stem cells

This invention is a divisional application of the invention patent with the application number "201710643072.1" and the invention title "a cell cryopreservation solution for human adipose stem cells".

technical field

The invention relates to the technical field of tissue cell culture, in particular to a human adipose stem cell cryopreservation solution and a preparation method and application thereof.

Background technique

Adipose-derived stem cells (ADSCs) are a kind of stem cells with multi-directional differentiation potential isolated from adipose tissue in recent years. They have the characteristics of general stem cells such as rapid expansion and not easy to age. Human adipose stem cells, namely human adipose-derived mesenchymal stem cells, are a kind of adult stem cells widely used in the field of tissue engineering and regenerative medicine, which have the same multi-directional differentiation potential as bone marrow mesenchymal stem cells. ADSCs showed fibroblast-like growth, abundant cytoplasm and nucleoli, arranged in parallel or swirl-like arrangement. Cell cycle analysis showed that 69% of cells were in G0/G1 phase, 24% in S phase, and 8% in G2/M phase. In the presence of fetal calf serum, the cells were subcultured for 2-3 days and the cells proliferated by 1 times. After multiple passages (10-20 generations), the cell proliferation rate did not slow down significantly, and senescent cells appeared in the cell population after 6 passages, and the senescent cells accounted for about 15% in the 15th generation cell population.

Due to the long period of isolation and culture of human adipose-derived mesenchymal stem cells, it takes about 2 weeks or more for the primary cells to reach a certain number, and it takes about 3 weeks to reach a certain number. Long-term culture and passage in vitro of human adipose-derived mesenchymal stem cells are prone to spontaneous differentiation and lose their multilineage differentiation potential. Therefore, in order to ensure the continuity of experiments, a large number of seed cells for experiments or tissue engineering must be provided at any time. Therefore, the demand for cells is very strong. Now, it is common practice to freeze the produced cells and thaw them several times before use. However, in the prior art, there are two types of cryopreservation solutions used for stem cell cryopreservation: serum-containing and serum-free. Since serum has the disadvantages of complex components, unstable quality, and high price, serum-free cryopreservation and resuscitation technology has become a development trend. The cryopreservation of stem cells mainly relies on the protection of antifreeze dimethyl sulfoxide (DMSO) and serum. Commonly used cell protectants include hydroxyethyl starch (ES), polyethylene glycol (PEG) and the like. However, DMSO can be toxic to cells and cause irreversible damage to cryopreserved stem cells. In order to obtain neural stem cells with convenient sources, it is an urgent need in this field to develop an effective cryopreservation method for neural stem cells. Establishing a method for long-term cryopreservation of human adipose stem cells is of great significance for promoting the research and application of human adipose stem cells.

Contents of the invention

The technical problem to be solved by the present invention is to provide a human adipose stem cell cryopreservation solution and the corresponding preparation method and use method.

Phoebe chinensis is a small tree. The applicant found through research that there are substances capable of cold resistance in the plant. Therefore, the applicant obtained the corresponding cold-resistant polypeptide through high-throughput screening. Therefore, the applicant tried It is added to the cryopreservation solution to preserve cells.

Technical scheme of the present invention is as follows:

A human fat stem cell cryopreservation solution is characterized in that it is composed of the following components: it is characterized in that the cryopreservation solution contains: 0.5% w/v low-density lipoprotein, 1% w/v trehalose, 1% glycerol , 1% w/v lecithin, peptide 0.1% w/v, bFGF 1% w/v, vitamin E 1% w/v, reduced glutathione 0.5% w/v, and finally use DMEM medium to dilute to 100ml, the sequence of the polypeptide is shown in any one of SEQ ID NO: 1-32. The polypeptide is obtained by the applicant through the screening of a large number of gene banks in the early stage, and the polypeptide has the function of protecting cells from damage. Its names are 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, which in turn correspond to SEQ ID NOs: 1-32.

In the cryopreservation solution of cells of the present invention, trehalose, vitamin E and polypeptides have a clear ability to resist damage to cells from external damage, especially polypeptides, and also protect cells from damage to cells caused by ice crystals during cryopreservation.

The present invention also provides a preparation method of the cryopreservation solution of human adipose stem cells, that is, mixing and shaking the components.

The present invention also provides a method for cryopreserving human adipose stem cells, comprising the following steps:

A. Preparation of cryopreservation solution: prepare the cell cryopreservation solution, and mix each component according to the corresponding ratio according to the conventional operation method to obtain;

B. Preparation of cell suspension: culture a bottle of human adipocytes grown into a monolayer, digest with 0.20% trypsin solution for 4 minutes, discard the trypsin solution, add 15ml of DMEM culture solution, blow gently with a pipette to make the cells uniform, and centrifuge for 4000 Spin/min, discard the supernatant, add 2ml of the cryopreservation solution in step A, mix well, and put it into a sterile cryopreservation tube;

C. Freezing: first freeze the cryopreservation tube at 4°C for 30min, then put it into -30°C for 1h, then put it into -80°C for 3h, and finally transfer it to liquid nitrogen for preservation;

D. Cell recovery: take out the cryopreservation tube and quickly place it in a 37°C water bath, shake until the cell suspension is completely melted, then dilute with 5 times DMEM, centrifuge at 1000r/min for 5min, remove the supernatant by centrifugation, and repeat 3 times. The cell suspension concentration is 10 ⁸ cells/ml-10 ¹⁰ cells/ml.

Beneficial effects of the present invention:

The human adipose stem cell cryopreservation solution of the present invention has a recovery cell survival rate of over 97%, which is significantly improved compared with the recovery survival rate of conventional cell cryopreservation solutions, and there is basically no loss of cells.

The stem cell cryopreservation solution of the invention can preserve the stem cells for a long time without changing the cell activity, thus ensuring the biological activity of the cells.

The nerve cell cryopreservation method of the present invention has simple and feasible operation, reasonable price and good practical value.

Detailed ways

The preparation of embodiment 1 polypeptide

Take 5g of the leaves of Phoebe chinensis, clean them, mince them, squeeze the juice, add papain and trypsin, the amount of enzymes added is 8000IU/g leaves, the enzymolysis temperature is 47°C, the pH value is 7.5, the enzymolysis time is 1.5h, and the enzymolysis After completion, the enzyme was extinguished at 90°C for 10 minutes; the insoluble matter was filtered to remove the insoluble matter after the enzyme was extinguished, and a solution was obtained. The obtained polypeptide solution was decolorized by adding 4% activated carbon adsorption, and the polypeptide was separated with Sephadex G-50 (Sephadex G-50), 20mmol/ Elute with L HCl solution, the flow rate is 1.3mL/min, collect the eluted products in different time periods, adjust the solution to pH 7.0, centrifuge at 10,000 rpm for 15 minutes, desalt with macroporous resin DA201-C, and concentrate in vacuo , and the supernatant was freeze-dried for later use; after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), small molecular weight bands were recovered, and after functional verification, a total of 32 small peptide sequences were obtained with stable Human adipose stem cells can promote the growth of stem cells and maintain the normal growth state of stem cells. According to different peak separation times in the chromatographic column, the corresponding small peptides can be obtained in batches, and the peptides can also be artificially synthesized. The sequence of the polypeptide is shown in SEQ ID NO: 1-32. They were named KATS-1~32 respectively.

Embodiment 2 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:1.

Example 3 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:2.

Example 4 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:3.

Example 5 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v, and finally use DMEM medium to make up the volume to 100ml, wherein the sequence of the polypeptide is shown in SEQ ID NO:4.

Example 6 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v, and finally use DMEM medium to make up the volume to 100ml, wherein the sequence of the polypeptide is shown in SEQ ID NO:5.

Example 7 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:6.

Example 8 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:7.

Example 9 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:8.

Example 10 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:9.

Example 11 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:10.

Example 12 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:11.

Example 13 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, 0.5% w/v of reduced glutathione, and finally use DMEM medium to make up the volume to 100ml, wherein the sequence of the polypeptide is shown in SEQ ID NO:12.

Example 14 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:13.

Example 15 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:14.

Example 16 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:15.

Example 17 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:16.

Example 18 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:17.

Example 19 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:18.

Example 20 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:19.

Example 21 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:20.

Example 22 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:21.

Example 23 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:22.

Example 24 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:23.

Example 25 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:24.

Example 26 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:25.

Example 27 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:26.

Example 28 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:27.

Example 29 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:28.

Example 30 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:29.

Example 31 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:30.

Example 32 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, reduced glutathione 0.5% w/v was finally adjusted to 100 ml with DMEM medium, wherein the sequence of the polypeptide is shown in SEQ ID NO:31.

Example 33 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v Trehalose, 1% Glycerin, 1% w/v Lecithin, Peptide 0.1% w/v, bFGF 1% w/v, Vitamin E 1% w/v, 0.5% w/v of reduced glutathione, and finally use DMEM medium to make up the volume to 100ml, wherein the sequence of the polypeptide is shown in SEQ ID NO:32.

Example 34 Preparation of Human Adipose Stem Cell Cryopreservation Solution

0.5% w/v LDL, 1% w/v trehalose, 1% glycerol, 1% w/v lecithin, bFGF 1% w/v, vitamin E 1% w/v, reduced glutathione Glycine 0.5% w/v was finally adjusted to 100ml with DMEM medium.

Example 35 Comparative example

The serum-free freezing solution authorized in CN 102550542B was used as the control freezing solution.

The effect verification of embodiment 36 cryopreservation solution

The cell cryopreservation solutions prepared in the above Examples 2-34 and Comparative Example were subjected to cell cryopreservation and recovery experiments according to the following methods.

Cell cryopreservation process:

Human adipose stem cells grown into a monolayer were cultured at a cell density of about 6*10 ⁹ cells/ml, and the cell surface was washed once by adding PBS with pH 7.0.

Digest the cells with 0.20% trypsin solution for 4 minutes, discard the trypsin solution, add 15 ml of DMEM culture solution, blow gently with a pipette to make the cells uniform, centrifuge at 4000 rpm, discard the supernatant, add Examples 1-33 and compare Mix 2ml of the cryopreservation solution prepared in Example 1, and put it into a sterile cryopreservation tube;

Freezing: first freeze the cryopreservation tubes at 4°C for 30 minutes, then store them at -30°C for 1 hour, then store them at -80°C for 3 hours, and finally transfer them to liquid nitrogen for storage; month, 8 months. 3 repetitions per set.

Cell recovery: take out the frozen tube and place it in a 37°C water bath quickly, shake until the cell suspension is completely melted, then dilute with 5 times DMEM, centrifuge at 1000r/min for 5min, remove the supernatant by centrifugation, repeat 3 times, and calculate the cell survival Rate. The result is as follows:

It can be seen from the above results that the cryopreservation solution of the present invention is particularly suitable for the cryopreservation culture of human adipose stem cells, and has better cell protection activity.

Example 37 Stem Cell Antigen Detection

Adipose stem cells have a variety of specific antigens and receptors, mainly 3, 13, D29, 34, 45, CD49e, CD59, CD73, CD90, CD105, HLA-ABC, etc.

Take the adipose stem cells frozen for 4 months in Example 36, remove the culture medium, mix and digest with 1:1 2.5% trypsin solution and 0.0296 EDTA solution, wash with PBS containing 1% bovine serum albumin (BSA) Make 1*1O6 single cell suspension per 100ul, divide into 7 equal parts, put into 7 Eppendorf tubes and number them respectively, add a total of 20μL of FITC Mouse IgGl, APC_CY7Mouse IgG2b and staining buffer to the first tube for detection As a control, add 20 μL each of CD29, CD34, 44, 45, 105, and HLA.DR monoclonal antibodies to other test tubes, and add 100 μL of cell suspension to each tube (containing 1*106 cells) , incubated at room temperature for 25 min, washed with PBS containing 1% BSA, and detected by flow cytometry. Analysis results: six surface antigens 29, 34, 44, CD45, CD105 and HLA.DR of human adipose stem cells were analyzed by flow cytometry, and the results showed that the cells cultured using the medium of Examples 2-34 had characteristics of human adipose stem cells. HLA.DR negative, rule out that these cells are fibroblasts.

Example 38 Adipogenic Induction and Detection

Since adipose stem cells have multi-directional differentiation ability, differentiation induction of adipose stem cells under certain conditions can obtain differentiated cells with specific functions.

Take the adipose stem cells frozen for 4 months in Example 36, add dexamethasone to the culture medium, and use the general method to induce adipogenicity of the adipose stem cells. It was found through culture that all the frozen stem cells could differentiate into adipocytes, and basically all of them differentiated into adipocytes with high activity. This fully demonstrates that the adipose stem cells after cryopreservation still retain the original cell activity.

What have been described above are only some embodiments of the present invention. Those of ordinary skill in the art can make some changes and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

sequence listing

<110> Li Qian

<120> A cell cryopreservation medium for human adipose stem cells

<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 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 (5)

1. A human adipose stem cell cryopreservation solution, characterized in that, is characterized in that it consists of the following components: 0.5% w/v low-density lipoprotein, 1% w/v trehalose, 1% glycerol, 1% w/v lecithin, polypeptide 0.1% w/v, bFGF 1% w/v, vitamin E 1% w/v, reduced glutathione 0.5% w/v, and finally use DMEM medium to make up to 100ml. 2. The preparation method of the human adipose stem cell cryopreservation solution according to claim 1, characterized in that it comprises mixing the components in the formula ratio to obtain the cryopreservation solution. 3. The application of the cryopreservation solution of human adipose stem cells according to claim 1 in improving the survival rate of frozen cells after resuscitation. 4. The cryopreservation solution of human adipose stem cells according to claim 1, wherein the polypeptide sequence is as shown in SEQ ID NO:5. 5. A polypeptide, characterized in that the sequence is as shown in SEQ ID NO:5.