CN114794083A - Cryopreservation liquid and cryopreservation method for stem cells and products thereof - Google Patents
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Images
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
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- 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 cryopreservation solution and a cryopreservation method for stem cells and products thereof. The frozen stock solution comprises the following raw materials in parts by weight: 10-15 parts of trehalose, 10-20 parts of EDTA (ethylene diamine tetraacetic acid), 5-8 parts of methyl cellulose, 3-5 parts of glycerol, 3-5 parts of dimethyl sulfoxide and 120 parts of a culture medium 100-. The frozen stock solution disclosed by the invention adopts trehalose, EDTA (ethylene diamine tetraacetic acid), methyl cellulose, glycerol, dimethyl sulfoxide and a culture medium, wherein the trehalose can be used as a protective component of the frozen stock solution, and the EDTA can provide a reducing environment for frozen cells; the methyl cellulose can improve the anti-apoptosis and anti-oxidation capability of the frozen stock solution after recovery; the glycerol and the dimethyl sulfoxide are matched for use, so that permeable components of the freezing solution can rapidly penetrate through cell membranes to enter cells, the freezing point is lowered, the freezing process is delayed, the ion concentration in the cells is improved, and the formation of ice crystals in the cells is reduced, so that the damage degree of the cells is reduced; reduces the potential toxic effect on cells.
Description
Technical Field
The invention relates to a cryopreservation solution for stem cells and products thereof and a cryopreservation method, and belongs to the technical field of cryopreservation solutions.
Background
Mesenchymal Stem Cells (MSCs) are derived from mesoderm tissues, belong to multipotent Stem Cells, are characterized by wide sources, low immunogenicity, certain differentiation potential, self-renewal capacity and immunity regulation capacity, and are mainly divided into human umbilical cord Mesenchymal Stem Cells, adipose Mesenchymal Stem Cells, bone marrow Mesenchymal Stem Cells, dental pulp Mesenchymal Stem Cells, placenta Mesenchymal Stem Cells and the like according to the sources.
At present, the cryopreservation solution of mesenchymal stem cells is mainly 5% -20% Fetal Bovine Serum (FBS) containing bovine serum components, a cell basal medium (Dulbecco's medium or alpha-MEM or F12/DMEM) and 10% DMSO. DMSO belongs to a cryoprotectant, can protect cells from being damaged by crystal substances in the cooling process, but the higher the content of DMSO is, the greater the toxic effect on the cells is, FBS introduces animal-derived components into human-derived cells, has certain potential safety hazards, such as introduction of mycoplasma and bovine-derived pathogenic bacteria, and has batch instability. If FBS is not added, the survival rate of the cells after recovery is greatly reduced. Therefore, in order to ensure the safety of mesenchymal stem cells and the survival rate after recovery, it is necessary to develop a frozen stock solution with clear components, no serum and high survival rate.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a cryopreservation solution and a cryopreservation method for stem cells and products thereof, wherein the cryopreservation solution does not contain serum, has definite components and higher survival rate.
The invention is realized by the following technical scheme:
the cryopreservation liquid for the stem cells and the products thereof comprises the following raw materials in parts by weight: 10-15 parts of trehalose, 10-20 parts of EDTA (ethylene diamine tetraacetic acid), 5-8 parts of methyl cellulose, 3-5 parts of glycerol, 3-5 parts of dimethyl sulfoxide and 120 parts of a culture medium 100-.
The culture medium of the freezing stock solution for the stem cells and the products thereof is a DMEM culture medium, a MEM culture medium, an IMEM culture medium or an RPMI1640 culture medium.
The cryopreservation liquid for the stem cells and the products thereof comprises glycerol and dimethyl sulfoxide in a weight ratio of 1: 1.
The cryopreservation method for the stem cells and the products thereof adopts the cryopreservation solution and comprises the following steps:
(1) preparing a freezing solution: under aseptic conditions, taking the raw materials according to the proportion, adding trehalose into a culture medium, mixing uniformly, then sequentially adding EDTA and methyl cellulose, mixing uniformly, adding glycerol and dimethyl sulfoxide, and fully mixing to obtain a frozen stock solution;
(2) stem cell treatment: taking out the stem cells from the culture bottle, adding pancreatin for digestion, centrifuging, removing the supernatant, adding a solution for cleaning, performing centrifugal separation again, removing the supernatant, adding a freezing medium for heavy suspension, and placing in a freezing tube or a freezing bag;
(3) freezing and storing: and (3) pre-freezing the freezing tube or the freezing bag in the step (2) at-18 to-22 ℃, and then freezing at-80 to-90 ℃.
In the cryopreservation method for the stem cells and the products thereof, in the step (1), the frozen stock solution is obtained after the raw materials are fully mixed and filtered and sterilized by using a filter.
In the cryopreservation method for the stem cells and the products thereof, in the step (2), the added solution is normal saline.
The cryopreservation method for the stem cells and the products thereof comprises the step (2) of controlling the concentration of the stem cells in the cryopreservation tube or the cryopreservation bag to be 4-6 multiplied by 10 6 Individual cells/mL.
The cryopreservation method for the stem cells and the products thereof has the advantages that the time for pre-cryopreservation is 10-20 min; then directly freezing and storing at-80 to-90 ℃.
The invention achieves the following beneficial effects:
the frozen stock solution disclosed by the invention adopts trehalose, EDTA (ethylene diamine tetraacetic acid), methyl cellulose, glycerol, dimethyl sulfoxide and a culture medium, wherein the trehalose can be used as a protective component of the frozen stock solution, and the EDTA can provide a reducing environment for frozen cells; the methylcellulose can improve the anti-apoptosis and anti-oxidation capability of the frozen stock solution after recovery; the glycerol and the dimethyl sulfoxide are matched for use, so that permeable components of the freezing solution can rapidly penetrate through cell membranes to enter cells, the freezing point is lowered, the freezing process is delayed, the ion concentration in the cells is improved, the formation of ice crystals in the cells is reduced, and meanwhile, the using amount of the dimethyl sulfoxide is reduced, so that the cell damage degree is reduced; the potential toxic effect on cells is reduced; in addition, the frozen stock solution does not contain FBS, so that the problem of animal-derived safety of the traditional frozen stock solution is solved.
The freezing solution is simple to prepare and low in cost; the cryopreservation method disclosed by the invention is simple in steps, and the cryopreservation liquid is effective for a long time through pre-cryopreservation.
Drawings
FIG. 1 is a whole cell image after 1 month of resuscitating culture using the frozen stock solution of example 1;
fig. 2 is an enlarged view of fig. 1.
FIG. 3 is a graph showing the proliferation of RTCA resuscitated after 1 week of cryopreservation using the cryopreservation solutions of example 1 and comparative example 5.
FIG. 4 is a graph showing the proliferation of RTCA resuscitated after 1 month of cryopreservation using the cryopreservation solutions of example 1 and comparative example 5.
Detailed Description
The invention is further described below. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Example 1
The cryopreservation liquid for the stem cells and the products thereof comprises the following raw materials in parts by weight: 10 parts of trehalose, 10 parts of EDTA (ethylene diamine tetraacetic acid), 5 parts of methyl cellulose, 3 parts of glycerol, 3 parts of dimethyl sulfoxide and 100 parts of MEM (MEM) culture medium.
The cryopreservation method for the stem cells and the products thereof adopts the cryopreservation solution and comprises the following steps:
(1) preparing a freezing solution: under the aseptic condition, taking the raw materials according to the proportion, adding trehalose into a culture medium, uniformly mixing, sequentially adding EDTA and methylcellulose, uniformly mixing, adding glycerol and dimethyl sulfoxide, fully mixing, and filtering and sterilizing by using a filter to obtain a frozen stock solution;
(2) stem cell treatment: taking out stem cells from the culture bottle, adding pancreatin for digestion, centrifuging, discarding the supernatant, adding physiological saline for cleaning, centrifuging again, discarding the supernatant,adding the freezing medium for re-suspension, and placing the mixture into a freezing tube or a freezing bag; the concentration of the stem cells in the cryopreservation tube or the cryopreservation bag is 4-6 multiplied by 10 6 Individual cells/mL;
(3) freezing and storing: and (3) pre-freezing the freezing tube or the freezing bag in the step (2) at-18 to-22 ℃ for 10-20min, and then freezing at-80 to-90 ℃.
Example 2
The cryopreservation liquid for the stem cells and the products thereof comprises the following raw materials in parts by weight: 15 parts of trehalose, 20 parts of EDTA (ethylene diamine tetraacetic acid), 8 parts of methyl cellulose, 5 parts of glycerol, 5 parts of dimethyl sulfoxide and 120 parts of DMEM (DMEM) culture medium.
The cryopreservation method for the stem cells and the products thereof adopts the cryopreservation solution and comprises the following steps:
(1) preparing a freezing solution: under the aseptic condition, taking the raw materials according to the proportion, adding trehalose into a culture medium, uniformly mixing, sequentially adding EDTA and methylcellulose, uniformly mixing, adding glycerol and dimethyl sulfoxide, fully mixing, and filtering and sterilizing by using a filter to obtain a frozen stock solution;
(2) stem cell treatment: taking out the stem cells from the culture bottle, adding pancreatin for digestion, centrifuging, removing the supernatant, adding physiological saline for cleaning, centrifuging again, removing the supernatant, adding a freezing medium for heavy suspension, and placing in a freezing tube or a freezing bag; the concentration of the stem cells in the cryopreservation tube or the cryopreservation bag is 4-6 multiplied by 10 6 Individual cells/mL;
(3) freezing and storing: and (3) pre-freezing the freezing tube or the freezing bag in the step (2) at-18 to-22 ℃ for 10-20min, and then freezing at-80 to-90 ℃.
Example 3
The cryopreservation liquid for the stem cells and the products thereof comprises the following raw materials in parts by weight: 12 parts of trehalose, 18 parts of EDTA (ethylene diamine tetraacetic acid), 6 parts of methyl cellulose, 4 parts of glycerol, 4 parts of dimethyl sulfoxide and 110 parts of IMEM (ethylene diamine tetraacetic acid) culture medium.
The cryopreservation method for the stem cells and the products thereof adopts the cryopreservation solution and comprises the following steps:
(1) preparing a freezing solution: under the aseptic condition, taking the raw materials according to the proportion, adding trehalose into a culture medium, uniformly mixing, sequentially adding EDTA and methylcellulose, uniformly mixing, adding glycerol and dimethyl sulfoxide, fully mixing, and filtering and sterilizing by using a filter to obtain a frozen stock solution;
(2) stem cell treatment: taking out the stem cells from the culture bottle, adding pancreatin for digestion, centrifuging, removing the supernatant, adding physiological saline for cleaning, centrifuging again, removing the supernatant, adding a freezing medium for heavy suspension, and placing in a freezing tube or a freezing bag; the concentration of the stem cells in the cryopreservation tube or cryopreservation bag is 4-6 multiplied by 10 6 Individual cells/mL;
(3) freezing and storing: and (3) pre-freezing the freezing tube or the freezing bag in the step (2) at-18 to-22 ℃ for 10-20min, and then freezing at-80 to-90 ℃.
Example 4
The cryopreservation liquid for the stem cells and the products thereof comprises the following raw materials in parts by weight: 13 parts of trehalose, 15 parts of EDTA (ethylene diamine tetraacetic acid), 7 parts of methyl cellulose, 4 parts of glycerol, 4 parts of dimethyl sulfoxide and 108 parts of RPMI1640 medium.
The cryopreservation method for the stem cells and the products thereof adopts the cryopreservation solution and comprises the following steps:
(1) preparing a freezing solution: under the aseptic condition, taking the raw materials according to the proportion, adding trehalose into a culture medium, uniformly mixing, sequentially adding EDTA and methylcellulose, uniformly mixing, adding glycerol and dimethyl sulfoxide, fully mixing, and filtering and sterilizing by using a filter to obtain a frozen stock solution;
(2) stem cell treatment: taking out the stem cells from the culture bottle, adding pancreatin for digestion, centrifuging, removing the supernatant, adding physiological saline for cleaning, centrifuging again, removing the supernatant, adding a freezing medium for heavy suspension, and placing in a freezing tube or a freezing bag; the concentration of the stem cells in the cryopreservation tube or the cryopreservation bag is 4-6 multiplied by 10 6 Individual cells/mL;
(3) freezing and storing: and (3) pre-freezing the freezing tube or the freezing bag in the step (2) at-18 to-22 ℃ for 10-20min, and then freezing at-80 to-90 ℃.
Comparative example 1
The cryopreservation liquid for the stem cells and the products thereof comprises the following raw materials in parts by weight: 10 parts of trehalose, 10 parts of EDTA (ethylene diamine tetraacetic acid), 5 parts of methyl cellulose, 6 parts of dimethyl sulfoxide and 100 parts of MEM (MEM) culture medium. The rest is the same as in example 1.
Comparative example 2
The cryopreservation liquid for the stem cells and the products thereof comprises the following raw materials in parts by weight: 10 parts of trehalose, 5 parts of methyl cellulose, 3 parts of glycerol, 3 parts of dimethyl sulfoxide and 100 parts of MEM culture medium. The rest is the same as in example 1.
Comparative example 3
The cryopreservation liquid for the stem cells and the products thereof comprises the following raw materials in parts by weight: 10 parts of trehalose, 10 parts of EDTA (ethylene diamine tetraacetic acid), 3 parts of glycerol, 3 parts of dimethyl sulfoxide and 100 parts of MEM (minimum essential oil minimum) culture medium. The rest is the same as in example 1.
Comparative example 4
The cryopreservation liquid for the stem cells and the products thereof comprises the following raw materials in parts by weight: 10 parts of trehalose, 10 parts of EDTA (ethylene diamine tetraacetic acid), 5 parts of methyl cellulose, 3 parts of glycerol, 3 parts of dimethyl sulfoxide and 100 parts of MEM (MEM) culture medium.
The cryopreservation method for the stem cells and the products thereof adopts the cryopreservation solution and comprises the following steps:
(1) preparing a freezing solution: under the aseptic condition, adding trehalose into a culture medium, mixing uniformly, then sequentially adding EDTA and methyl cellulose, mixing uniformly, adding glycerol and dimethyl sulfoxide, fully mixing, and then filtering and sterilizing by using a filter to obtain a frozen stock solution;
(2) stem cell treatment: taking out the stem cells from the culture bottle, taking the raw materials according to the proportion, adding pancreatin for digestion, centrifuging, removing the supernatant, adding physiological saline for cleaning, centrifuging again, removing the supernatant, adding a freezing medium for heavy suspension, and placing in a freezing tube or a freezing bag; the concentration of the stem cells in the cryopreservation tube or the cryopreservation bag is 4-6 multiplied by 10 6 Individual cells/mL;
(3) freezing and storing: and (3) freezing and storing the freezing and storing tube or the freezing and storing bag in the step (2) at the temperature of between 80 ℃ below zero and 90 ℃ below zero.
Comparative example 5
The cryopreservation liquid for the stem cells and the products thereof comprises the following raw materials in parts by weight: 10 parts of fetal bovine serum, 3 parts of glycerol, 3 parts of dimethyl sulfoxide and 100 parts of MEM (minimum organic Membrane) culture medium. The rest is the same as in example 1.
Dividing human umbilical cord mesenchymal stem cells into 27 groups, respectively using the cryopreservation solution provided by the embodiments 1-4 and the comparative examples 1-5 of the invention to cryopreserve the human umbilical cord mesenchymal stem cells for 1 week, 1 month and 4 months, then recovering the cells, and detecting the survival rate of the recovered mesenchymal stem cells, wherein the results are shown in table 1:
TABLE 1 examples and comparative examples the survival rates of cells after cryopreservation and recovery
As can be seen from table 1, compared to comparative example 1, the survival rate of cells in 1 week of cryopreservation is comparable to that of cells in example 1 in which glycerol is added instead of part of dmso, and even better after 4 months of cryopreservation, so that it is feasible to replace part of dmso with glycerol, and the potential toxic effect on cells can be reduced; compared with the comparative example 2, the EDTA is added in the embodiment 1, so that the survival rate of the frozen stock solution after cell recovery can be further improved; compared with the comparative example 3, the methyl cellulose is added in the embodiment 1, so that the survival rate of the frozen stock solution after cell recovery can be further improved; compared with the comparative example 4, the pre-freezing storage is carried out in the example 1, so that the freezing storage liquid provided by the invention has better freezing storage effect under the condition of long freezing storage time.
Meanwhile, the cryopreserved cells of example 1 were taken out and cultured for 1 month. The whole cells after 1 month of culture are shown in FIG. 1, and the morphology after amplification is shown in FIG. 2. Therefore, the mesenchymal stem cells have the advantages of remarkably improving the survival rate and having good cell state by using the cryopreservation solution.
Detecting the activity of the cells after the umbilical cord mesenchymal stem cells are recovered: and (3) detecting the proliferation capacity of the cells when the stem cells are recovered after being frozen for different time by using RTCA (real-time marker-free cell analysis system). The results of the RTCA are shown in the figure, and figure 3 is a graph of the proliferation of the recovered RTCA after the umbilical cord mesenchymal stem cells are frozen for 1 week by using the frozen stock solutions of example 1 and comparative example 5; FIG. 4 is a graph showing the proliferation of RTCA resuscitated after cryopreservation of umbilical cord mesenchymal stem cells for 1 month using the cryopreservation solutions of example 1 and comparative example 5. As can be seen from FIGS. 3 and 4, the cryopreservation solution of the invention is suitable for the preservation of umbilical cord mesenchymal stem cells, and does not affect the proliferation capacity of the umbilical cord mesenchymal stem cells. And because the concentration of the dimethyl sulfoxide is low, the toxicity to other cells in the body is small after the dimethyl sulfoxide is directly injected.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The cryopreservation liquid for the stem cells and the products thereof is characterized by comprising the following raw materials in parts by weight: 10-15 parts of trehalose, 10-20 parts of EDTA (ethylene diamine tetraacetic acid), 5-8 parts of methyl cellulose, 3-5 parts of glycerol, 3-5 parts of dimethyl sulfoxide and 120 parts of a culture medium 100-.
2. The cryopreservation solution for stem cells and products thereof according to claim 1, wherein the culture medium is DMEM medium, MEM medium, IMEM medium or RPMI1640 medium.
3. The cryopreservation solution for stem cells and products thereof according to claim 1, wherein the weight ratio of glycerol to dimethyl sulfoxide is 1: 1.
4. The method for freezing and preserving stem cells and products thereof by using the freezing and preserving solution as claimed in any one of claims 1 to 3, which is characterized by comprising the following steps:
(1) preparing a freezing solution: under the aseptic condition, taking the raw materials according to the proportion, adding trehalose into a culture medium, uniformly mixing, sequentially adding EDTA and methylcellulose, uniformly mixing, adding glycerol and dimethyl sulfoxide, and fully mixing to obtain a frozen stock solution;
(2) stem cell treatment: taking out the stem cells from the culture bottle, adding pancreatin for digestion, centrifuging, removing the supernatant, adding a solution for cleaning, performing centrifugal separation again, removing the supernatant, adding a freezing medium for heavy suspension, and placing in a freezing tube or a freezing bag;
(3) freezing and storing: and (3) pre-freezing the freezing tube or the freezing bag in the step (2) at-18 to-22 ℃, and then freezing at-80 to-90 ℃.
5. The cryopreservation method for stem cells and products thereof according to claim 4, wherein in the step (1), the frozen stock solution is obtained by thoroughly mixing the raw materials, and then performing filtration sterilization by using a filter.
6. The cryopreservation method for stem cells and products thereof according to claim 4, wherein in the step (2), the added solution is physiological saline.
7. The cryopreservation method for stem cells and products thereof according to claim 4, wherein the concentration of stem cells in the cryopreservation tube or cryopreservation bag in the step (2) is 4-6 x 10 6 Individual cells/mL.
8. The cryopreservation method for stem cells and products thereof according to claim 4, wherein the time for pre-cryopreservation is 10-20 min; then directly freezing and storing at-80 to-90 ℃.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210608510.1A CN114794083A (en) | 2022-05-31 | 2022-05-31 | Cryopreservation liquid and cryopreservation method for stem cells and products thereof |
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| EP3039124A1 (en) * | 2013-08-29 | 2016-07-06 | Stempeutics Research Private Limited | Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof |
| CN110338187A (en) * | 2018-04-08 | 2019-10-18 | 生物角（厦门）科技有限公司 | A kind of mesenchymal stem cell serum-free frozen stock solution composition |
| CN110946129A (en) * | 2019-11-08 | 2020-04-03 | 浙江卫未生物医药科技有限公司 | High-survival-rate frozen stock solution after cell recovery |
| CN114190368A (en) * | 2022-01-06 | 2022-03-18 | 上海合佑生生物科技有限公司 | Serum-free immune cell cryopreservation liquid, preparation method and immune cell cryopreservation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3039124A1 (en) * | 2013-08-29 | 2016-07-06 | Stempeutics Research Private Limited | Stromal cells derived conditioned medium, method of obtaining said conditioned medium compositions, formulations and applications thereof |
| CN110338187A (en) * | 2018-04-08 | 2019-10-18 | 生物角（厦门）科技有限公司 | A kind of mesenchymal stem cell serum-free frozen stock solution composition |
| CN110946129A (en) * | 2019-11-08 | 2020-04-03 | 浙江卫未生物医药科技有限公司 | High-survival-rate frozen stock solution after cell recovery |
| CN114190368A (en) * | 2022-01-06 | 2022-03-18 | 上海合佑生生物科技有限公司 | Serum-free immune cell cryopreservation liquid, preparation method and immune cell cryopreservation method |
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