CN114451402A - Adipose-derived mesenchymal stem cell preservation solution and preparation method and application thereof - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Abstract
The invention discloses a fat mesenchymal stem cell preservation solution which comprises the following components: basic culture medium, trehalose, coriolus versicolor glycopeptide, hydroxyethyl starch, horse chestnut seed extract, adenosine and L-glutamine. The preservation solution provided by the invention realizes the preservation of the adipose-derived mesenchymal stem cells at 4 ℃, the survival rate of the cells is more than 90% after the adipose-derived mesenchymal stem cells are preserved for 72 hours, the preservation requirement of clinic is fully met, the requirement on the preservation condition of the cells is reduced, the preservation solution can be adopted for the adipose-derived mesenchymal stem cells needing short-distance transportation, and the cost of preservation and transportation is reduced. The invention also provides a preparation method of the adipose-derived mesenchymal stem cell preservation solution, which is simple in process and easy to operate. The invention also provides a specific application of the preservation solution, and when the preservation solution is used for preserving the adipose mesenchymal stem cells, the cell activity is basically not influenced.
Description
Technical Field
The invention relates to the field of stem cells, in particular to a fat mesenchymal stem cell preservation solution and a preparation method and application thereof.
Background
Mesenchymal stem cells (MSC, mesnchymal stem cells) are important members of the stem cell family, are derived from mesoderm and ectoderm in early development, belong to pluripotent stem cells, are found in bone marrow, cord blood and fat, are pluripotent stem cells, and are concerned by people due to the multidirectional differentiation potential. The MSCs have wide clinical application prospect and are the first choice seed cells for cell replacement therapy and tissue engineering.
Adipose-derived stem cells (ADSCs) are a kind of stem cells having a multi-directional differentiation potential separated from adipose tissues in recent years, and can be differentiated into osteoblasts, chondrocyte adipocytes, and the like. The adipose-derived mesenchymal stem cells have many advantages, such as wide sources, easy separation and acquisition, no ethical problem and the like, and become a hot spot for clinical research and application in recent years.
Because of limited quantity, the adipose tissue-derived mesenchymal stem cells directly separated by self-body can not meet the clinical application requirements, the adipose tissue-derived mesenchymal stem cells obtained by separation need to be amplified in vitro, the in vitro culture period of the adipose tissue-derived mesenchymal stem cells is long, the cells with enough quantity can be obtained by 3-4 weeks of culture time, and the cells obtained by culture sometimes face the condition of short-time storage. The existing preservation method is to preserve in liquid nitrogen under the condition of adding a cryopreservation protective agent, and the liquid nitrogen is adopted to preserve cells, so that the requirements on the operation process and the cryopreservation equipment are higher, the cells can be damaged, and the activity of the cells is influenced. The preservation cost is increased for cells requiring temporary preservation for a short time.
Therefore, it is necessary to research a preservation solution, which can preserve cells at normal temperature while ensuring cell activity, and provide guarantee for temporary preservation of adipose-derived mesenchymal stem cells.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide the adipose-derived mesenchymal stem cell preservation solution, which can realize the preservation of adipose-derived mesenchymal stem cells at 4 ℃, and the activity of the preserved adipose-derived mesenchymal stem cells is not affected.
The second purpose of the invention is to provide a preparation method of the adipose-derived mesenchymal stem cell preservation solution.
The invention also aims to provide an application of the adipose mesenchymal stem cell preservation solution.
One of the purposes of the invention is realized by adopting the following technical scheme:
a preserving fluid for adipose-derived mesenchymal stem cells comprises the following components: basic culture medium, trehalose, coriolus versicolor glycopeptide, hydroxyethyl starch, horse chestnut seed extract, adenosine and L-glutamine.
Preferably, the basal medium is DMEM/F12 medium.
Preferably, the final concentration of each component in the medium is: trehalose 15-20 μ g/mL, coriolus versicolor glycopeptide 9-13 μ g/mL, hydroxyethyl starch 1-5mg/mL, horse chestnut seed extract 3-7 μ g/mL, adenosine 10-15 μ g/mL, L-glutamine 8-14 μ g/mL.
Preferably, the final concentration of each component in the medium is: trehalose 17 μ g/mL, coriolus versicolor glycopeptide 10 μ g/mL, hydroxyethyl starch 3mg/mL, horse chestnut seed extract 5 μ g/mL, adenosine 12 μ g/mL, L-glutamine 12 μ g/mL.
The second purpose of the invention is realized by adopting the following technical scheme:
the preparation method of the adipose tissue-derived stem cell preservation solution comprises the following steps: adding trehalose, corious versicolor glycopeptide, semen Aesculi extract, hydroxyethyl starch, adenosine, and L-glutamine into basic culture medium, stirring, mixing, filtering, sterilizing, and storing at 4-6 deg.C.
Preferably, the bacteria are removed by filtration through a 0.22 μm microfiltration membrane.
The third purpose of the invention is realized by adopting the following technical scheme:
an application of the preservation solution for the adipose tissue-derived stem cells, which is to resuspend the adipose tissue-derived stem cells in the preservation solution and preserve the adipose tissue-derived stem cells at 4 ℃.
Preferably, the density of adipose-derived mesenchymal stem cells in the preservation solution is 2-5X 106one/mL.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a preservation solution for adipose-derived mesenchymal stem cells, which realizes the preservation of adipose-derived mesenchymal stem cells at 4 ℃, ensures that the survival rate of the adipose-derived mesenchymal stem cells is more than 90% after the adipose-derived mesenchymal stem cells are preserved for 72 hours, and fully meets the clinical preservation requirement.
The invention also provides a preparation method of the adipose-derived mesenchymal stem cell preservation solution, which is simple in process and easy to operate. The invention also provides the specific application of the preservation solution, and the cell activity is basically not influenced when the preservation solution is used for preserving the adipose mesenchymal stem cells.
Drawings
Fig. 1 shows the survival rates of cells according to the change of the preservation time when the preservation solutions of examples 1 to 3 and comparative examples 1 to 5 of the present invention are used for preserving adipose mesenchymal stem cells.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
Example 1
A preserving fluid for adipose-derived mesenchymal stem cells consists of a DMEM/F12 culture medium and the following components added in the culture medium: trehalose, polysaccharopeptide, hydroxyethyl starch, horse chestnut seed extract, adenosine and L-glutamine, wherein the final concentration of each component in a culture medium is as follows: trehalose 17 μ g/mL, coriolus versicolor glycopeptide 10 μ g/mL, hydroxyethyl starch 3mg/mL, horse chestnut seed extract 5 μ g/mL, adenosine 12 μ g/mL, L-glutamine 12 μ g/mL.
A preparation method of adipose-derived mesenchymal stem cell preservation solution comprises the following steps: adding trehalose, corious versicolor glycopeptide, horse chestnut seed extract, hydroxyethyl starch, adenosine, and L-glutamine into DMEM/F12 culture medium, stirring, mixing, filtering with 0.22 μm microporous membrane for sterilization, and storing at 4 deg.C.
Example 2
A preserving fluid for adipose-derived mesenchymal stem cells consists of a DMEM/F12 culture medium and the following components added in the culture medium: trehalose, polysaccharopeptide, hydroxyethyl starch, horse chestnut seed extract, adenosine and L-glutamine, wherein the final concentration of each component in a culture medium is as follows: the final concentration of each component in the culture medium is: trehalose 15 μ g/mL, coriolus versicolor glycopeptide 9 μ g/mL, hydroxyethyl starch 1mg/mL, horse chestnut seed extract 3 μ g/mL, adenosine 10 μ g/mL, L-glutamine 8 μ g/mL.
A preparation method of adipose-derived mesenchymal stem cell preservation solution comprises the following steps: adding trehalose, corious versicolor glycopeptide, horse chestnut seed extract, hydroxyethyl starch, adenosine, and L-glutamine into DMEM/F12 culture medium, stirring, mixing, filtering with 0.22 μm microporous membrane for sterilization, and storing at 6 deg.C.
Example 3
A preserving fluid for adipose-derived mesenchymal stem cells consists of a DMEM/F12 culture medium and the following components added in the culture medium: trehalose, polysaccharopeptide, hydroxyethyl starch, horse chestnut seed extract, adenosine and L-glutamine, wherein the final concentration of each component in a culture medium is as follows: the final concentration of each component in the culture medium is: trehalose 20 μ g/mL, coriolus versicolor glycopeptide 13 μ g/mL, hydroxyethyl starch 5mg/mL, horse chestnut seed extract 7 μ g/mL, adenosine 15 μ g/mL, L-glutamine 14 μ g/mL.
A preparation method of adipose-derived mesenchymal stem cell preservation solution comprises the following steps: adding trehalose, corious versicolor glycopeptide, horse chestnut seed extract, hydroxyethyl starch, adenosine, and L-glutamine into DMEM/F12 culture medium, stirring, mixing, filtering with 0.22 μm microporous membrane for sterilization, and storing at 4 deg.C.
Comparative example 1
Comparative example 1 provides an adipose-derived mesenchymal stem cell preservation solution, which is distinguished from example 1 in that: the glycopeptides of corious versicolor and the extract of horse chestnut seeds were omitted and the procedure was as in example 1.
Comparative example 2
Comparative example 2 provides an adipose-derived mesenchymal stem cell preservation solution, which is distinguished from example 1 in that: the glycopeptides of corious versicolor are omitted, and the procedure is as described in example 1.
Comparative example 3
Comparative example 3 provides an adipose-derived mesenchymal stem cell preservation solution, which is distinguished from example 1 in that: the horse chestnut seed extract was omitted and the rest was the same as in example 1.
Comparative example 4
Comparative example 4 provides an adipose-derived mesenchymal stem cell preservation solution, which is distinguished from example 1 in that: the horse chestnut seed extract was omitted, and the amount of corious versicolor glycopeptide was adjusted to 15. mu.g/mL, the rest being the same as in example 1.
Comparative example 5
Comparative example 5 provides an adipose-derived mesenchymal stem cell preservation solution, which is distinguished from example 1 in that: the glycopeptide of corious versicolor is omitted, and the amount of the extract of the seed of the horse chestnut is adjusted to 15 μ g/mL, the rest being the same as in example 1.
Test examples
Washing adipose tissue with PBS (equal volume), centrifuging, collecting the last adipose tissue, washing for three times, adding collagenase I0.25% into the adipose tissue, digesting for 30min, centrifuging, adding DMEM/F12 culture medium containing 10% FBS to resuspend cells, inoculating into T75 culture bottle, culturing at 37 deg.C and 5% CO2The culture box is used for culturing, after the cell confluence is 80%, 0.25% of pancreatin is added for digestion, subculture is carried out, and the 3 rd generation adipose-derived mesenchymal stem cells obtained by subculture are taken for storage experiment.
The adipose-derived mesenchymal stem cells obtained by the above culture are resuspended by the preservation solution of examples 1 to 3 and comparative examples 1 to 5, and are subpackaged in freezing tubes with 1mL per tube and the cell density of 3X 106The cells/mL are stored at 4 ℃, sampled every 24 hours, and subjected to trypan blue staining to detect the survival rate of the cells, and a full-automatic cell counter Countstar is used to detect the cell clustering rate, wherein the cell survival rate result is shown in a figure 1, and the cell clustering rate result is shown in a table 1.
TABLE 1
It can be seen from table 1 that the cell-clumping rate increases with the increase of the storage time, wherein the cell-clumping rate is lower in examples 1 to 3 than in comparative examples 1 to 5. In comparative examples 1 to 5, one or more of polysaccharopeptide and horse chestnut seed extract are respectively omitted, and the cell agglomeration rate is higher than that in example 1 no matter whether the amount of the residual components is adjusted, which shows that the preservation solution of the invention can reduce the agglomeration rate of the adipose-derived mesenchymal stem cells in the preservation process and prolong the preservation time of the cells.
It can be seen from FIG. 1 that the survival rate of the cells decreased with the increase of the preservation time, but the survival rate of the cells decreased to different degrees in different preservation solutions. The survival rate of the cells in the preservation solution of the examples 1 to 3 is over 90 percent after the cells are preserved for 72 hours at 4 ℃, and the survival rate of the cells is over 84 percent after the cells are preserved for 96 hours, thereby fully meeting the clinical use requirement. In comparative example 1, polysaccharopeptide and horse chestnut seed extract are omitted, and the cell survival rate is only 67.49% after the cells are stored for 72h, which is remarkably reduced. The glycopeptides of coriolus versicolor and the extracts of horse chestnut seeds in comparative example 2 and comparative example 3 were omitted, and the cell survival rates after 72 hours of storage were 81.58% and 78.28%, respectively, and after 96 hours of storage were only 71.61% and 67.05%, respectively, which are far from example 1. In comparative example 4 and comparative example 5, after the polysaccharopeptide and the horse chestnut seed extract are respectively omitted, the using amount of the residual components is increased, the survival rate of the cells after being stored for 72 hours is 75.44 percent and 78.92 percent respectively, and the survival rate of the cells after being stored for 96 hours is only 64.64 percent and 66.45 percent respectively, so that the clinical requirement on the cell activity cannot be met.
The cells after 72h of storage in example 1 were collected, returned to room temperature, centrifuged to remove the storage medium, and the cells were resuspended in 10% FBS-containing DMEM/F12 medium at a cell density of 1X 104and/mL, inoculating into 12-well plates, culturing for 7 days continuously at 1mL per well, counting the number of the cultured cells, and using the non-preserved generation 3 adipose-derived mesenchymal stem cells as a control group, wherein the results are shown in table 2.
TABLE 2
| Group of | Number of cells (. times.10)4One) |
| Example 1 | 34.95 |
| Control group | 36.79 |
As can be seen from table 2, the difference between the proliferation activity of the adipose-derived mesenchymal stem cells preserved in example 1 and the cells that are not preserved is not great, and thus it can be seen that the preservation solution of the present invention can preserve adipose-derived mesenchymal stem cells at 4 ℃ for a certain period of time, and the cell survival rate and the cell proliferation activity are not substantially affected during the preservation process, and is suitable for the short-time preservation of adipose-derived mesenchymal stem cells.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (8)
1. The adipose-derived mesenchymal stem cell preservation solution is characterized by comprising the following components: basic culture medium, trehalose, coriolus versicolor glycopeptide, hydroxyethyl starch, horse chestnut seed extract, adenosine and L-glutamine.
2. The adipose-derived mesenchymal stem cell preservation solution according to claim 1, wherein the basic culture medium is DMEM/F12 culture medium.
3. The adipose-derived mesenchymal stem cell preservation solution according to claim 1, wherein the final concentration of each component in the culture medium is as follows: trehalose 15-20 μ g/mL, coriolus versicolor glycopeptide 9-13 μ g/mL, hydroxyethyl starch 1-5mg/mL, horse chestnut seed extract 3-7 μ g/mL, adenosine 10-15 μ g/mL, L-glutamine 8-14 μ g/mL.
4. The adipose-derived mesenchymal stem cell preservation solution according to claim 3, wherein the final concentration of each component in the culture medium is as follows: trehalose 17 μ g/mL, coriolus versicolor glycopeptide 10 μ g/mL, hydroxyethyl starch 3mg/mL, horse chestnut seed extract 5 μ g/mL, adenosine 12 μ g/mL, L-glutamine 12 μ g/.
5. The method for preparing the adipose-derived mesenchymal stem cell preservation fluid according to any one of claims 1 to 4, which comprises the following steps: adding trehalose, corious versicolor glycopeptide, semen Aesculi extract, hydroxyethyl starch, adenosine, and L-glutamine into basic culture medium, stirring, mixing, filtering, sterilizing, and storing at 4-6 deg.C.
6. The method for preparing the adipose-derived mesenchymal stem cell preservation solution according to claim 5, wherein the filtration sterilization is performed by using a 0.22 μm microporous filter membrane.
7. The application of the adipose tissue-derived stem cell preservation solution is characterized in that adipose tissue-derived stem cells are resuspended in the preservation solution and preserved at 4 ℃.
8. The use of the preservation solution for adipose-derived mesenchymal stem cells according to claim 7, wherein the density of adipose-derived mesenchymal stem cells in the preservation solution is 2-5 x 106one/mL.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115176796A (en) * | 2022-07-12 | 2022-10-14 | 广州沙艾生物科技有限公司 | Adipose-derived mesenchymal stem cell storage solution |
| CN116965403A (en) * | 2023-09-22 | 2023-10-31 | 诺莱生物医学科技有限公司 | Stem cell preservation solution and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107912421A (en) * | 2017-11-20 | 2018-04-17 | 温州医科大学附属第医院 | A kind of stem cell preserving fluid for Bone Marrow Mesenchymal Stem Cells Transplantation |
| US20210084891A1 (en) * | 2016-02-17 | 2021-03-25 | Japan Advanced Institute Of Science And Technology | Vitreous state stabilizing agent for animal cell cryopreservation solution |
| CN113854280A (en) * | 2021-09-06 | 2021-12-31 | 创芯国际生物科技(广州)有限公司 | Novel low-temperature preservation solution and preparation method and application thereof |
| CN113925049A (en) * | 2021-12-17 | 2022-01-14 | 广东乾晖生物科技有限公司 | Cell preservation solution for maintaining cell activity and preparation method and application thereof |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210084891A1 (en) * | 2016-02-17 | 2021-03-25 | Japan Advanced Institute Of Science And Technology | Vitreous state stabilizing agent for animal cell cryopreservation solution |
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