CN112970743B - Adipose-derived stem cell cryopreservation liquid and application thereof - Google Patents
Adipose-derived stem cell cryopreservation liquid and application thereof Download PDFInfo
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- CN112970743B CN112970743B CN202110243571.8A CN202110243571A CN112970743B CN 112970743 B CN112970743 B CN 112970743B CN 202110243571 A CN202110243571 A CN 202110243571A CN 112970743 B CN112970743 B CN 112970743B
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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
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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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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Abstract
The invention provides an adipose-derived stem cell and application thereof, and belongs to the technical field of stem cell cryopreservation. The fat stem cell freezing medium provided by the invention comprises 40% DMEM/F12, 50% FBS, 10% DMSO and 100-250 mu M rana japonica peptide. The adipose-derived stem cells frozen for a long time by using the freezing medium provided by the invention have better activity and adipogenic transformation capacity, and the apoptosis of the adipose-derived stem cells frozen for a long time can be effectively reduced by using the freezing medium provided by the invention.
Description
Technical Field
The invention belongs to the technical field of stem cell cryopreservation, and particularly relates to an adipose-derived stem cell cryopreservation solution and application thereof.
Background
Stem cell therapy is a novel therapeutic modality combining biomaterial science and tissue engineering technologies. Common stem cells include: bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells and peripheral blood mesenchymal stem cells. Adipose-derived mesenchymal stem cells are an ideal source of stem cells in tissue engineering because of being more easily obtained. Research shows that the adipose-derived mesenchymal stem cells not only have strong proliferation and differentiation capacities, but also can differentiate in the directions of fat, bone, cartilage, nerve, muscle and the like. At present, the recovery efficiency of adipose-derived mesenchymal stem cells is generally low, and the clinical transformation and basic research of adipose-derived mesenchymal stem cells are seriously restricted by the problem. Therefore, the preparation of the effective cryopreservation solution for reducing the damage of the adipose-derived mesenchymal stem cells in the cryopreservation process has important significance for the adipose-derived stem cell treatment.
Disclosure of Invention
The invention aims to provide an adipose-derived stem cell cryopreservation solution and application thereof.
In order to achieve the aim, the invention provides an adipose-derived stem cell cryopreservation solution, which comprises the following components: 40% DMEM/F12, 50% FBS, 10% DMSO, and anoplosin.
Preferably, the cryopreservation liquid comprises the following components: 40% DMEM/F12, 50% FBS, 10% DMSO, 100-.
Preferably, the cryopreservation liquid comprises the following components: 40% DMEM/F12, 50% FBS, 10% DMSO, 200 μ M peptide of Rana temporaria.
Preferably, the amino acid sequence of the peptide of the rana amurensis is Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-Val-Gly-Leu-Met-NH 2.
Secondly, the invention provides a polypeptide for adipose-derived stem cell cryopreservation liquid, wherein the polypeptide is the peptide of the rana amurensis.
Preferably, the rana amurensis peptide is used for maintaining the activity of the adipose-derived stem cells in the adipose-derived stem cell freezing solution;
the rana amurensis peptide is used for reducing apoptosis of adipose-derived stem cells in the adipose-derived stem cell cryopreservation liquid;
the rana japonica peptide is used for maintaining the adipogenic differentiation capacity of the adipose-derived stem cells in the adipose-derived stem cell cryopreservation liquid.
Secondly, the invention provides the application of the rana japonica peptide in preparing the maintenance agent of the activity of the adipose-derived stem cells.
Secondly, the invention provides the application of the rana japonica peptide in preparing the adipose-derived stem cell apoptosis inhibitor.
Secondly, the invention provides the application of the rana japonica peptide in preparing a maintenance agent for adipogenic differentiation of adipose-derived stem cells.
Preferably, the amino acid sequence of the peptide of the rana amurensis is Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-Val-Gly-Leu-Met-NH 2.
The invention has the beneficial effects that:
the adipose-derived stem cell cryopreservation solution provided by the invention can effectively maintain the activity of adipose-derived stem cells and reduce the apoptosis of the adipose-derived stem cells under a long-term cryopreservation environment, and meanwhile, the adipose-derived stem cells frozen for a long term by using the cryopreservation solution can keep better adipogenic transformation capacity.
Drawings
FIG. 1 expression levels of Cleaved-Caspase3 and Cleaved-Caspase9 in cryopreserved cells of example 1 and example 5;
FIG. 2 Lipidation transformation ability of cryopreserved cells in examples 1 and 5.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1
(1) The frozen stock solution A is prepared according to the following formula: 40% DMEM/F12, 50% FBS, 10% DMSO;
(2) will be 1 × 106The human adipose-derived mesenchymal stem cells are mixed with 1ml of the cryopreservation solution A, placed in a cryopreservation box at-80 ℃ for overnight, and then transferred to liquid nitrogen for preservation.
Example 2
(1) The frozen stock solution B is prepared according to the following formula: 40% DMEM/F12, 50% FBS, 10% DMSO, 50 μ M ranotide;
(2) will be 1 × 106The human adipose-derived mesenchymal stem cells are mixed with 1ml of the cryopreservation solution B, placed in a cryopreservation box at-80 ℃ for overnight, and then transferred to liquid nitrogen for preservation.
Example 3
(1) The frozen stock solution C is prepared according to the following formula: 40% DMEM/F12, 50% FBS, 10% DMSO, 100 μ M ranotide;
(2) will be 1 × 106The human adipose-derived mesenchymal stem cells were mixed with 1ml of the cryopreservation solution C, placed in a cryopreservation box at-80 ℃ overnight, and then transferred to liquid nitrogen for preservation.
Example 4
(1) The frozen stock solution D is prepared according to the following formula: 40% DMEM/F12, 50% FBS, 10% DMSO, 150 μ M ranotide;
(2) will be 1 × 106The human adipose-derived mesenchymal stem cells were mixed with 1ml of the cryopreservation solution D, placed in a cryopreservation box at-80 ℃ overnight, and then transferred to liquid nitrogen for preservation.
Example 5
(1) The frozen stock solution E is prepared according to the following formula: 40% DMEM/F12, 50% FBS, 10% DMSO, 200 μ M ranotide;
(2) will be 1 × 106The human adipose-derived mesenchymal stem cells were mixed with 1ml of the cryopreservation solution D, placed in a cryopreservation box at-80 ℃ overnight, and then transferred to liquid nitrogen for preservation.
Example 6
(1) The frozen stock solution F is prepared according to the following formula: 40% DMEM/F12, 50% FBS, 10% DMSO, 250 μ M ranotide;
(2) will be 1 × 106The human adipose-derived mesenchymal stem cells were mixed with 1ml of the cryopreservation solution D, placed in a cryopreservation box at-80 ℃ overnight, and then transferred to liquid nitrogen for preservation.
Experimental example 1
1. Cell viability assay
(1) After the cells of examples 1-6 were frozen for 12 months, the cells were thawed in a 37 ℃ water bath;
(2) 100 mul of the revived cells were taken and put into a 1.5ml centrifuge tube, 100 mul of the phenol blue staining solution was added for staining for 5min, 10 mul of the cell stain was aspirated and counted on a blood counting chamber, and the cell viability was calculated and the experiment was repeated 3 times per group.
Cell viability of cryopreserved cells for each example is as follows:
as can be seen from the above table, the cell viability was higher in examples 2-6 than in example 1, but the P value was greater than 0.05 in example 2, and thus it was not statistically significant. Therefore, when 100-250 mu M of the anoplophora peptide is added into the frozen stock solution in the prior art, the activity of the frozen cells can be obviously improved. Although the survival rate of the cryopreserved cells in example 6 is improved to a certain extent compared with that of the cryopreserved cells in example 5, the improvement is not statistically significant, and therefore, 200 mu M of the rana japonica peptide is taken as the optimal concentration according to the invention based on the principle of conservation.
Example 2
Apoptosis-related protein detection
(1) After the cells of example 1 and example 5 were frozen for 12 months, the cells were thawed in a 37 ℃ water bath;
(2) after cells are collected by centrifugation, adding a protein lysate containing a protease inhibitor and a phosphatase inhibitor to lyse the cells;
(3) after cracking on ice for 30min, transferring the protein lysate into an EP tube, and centrifuging for 10min by a low-temperature high-speed centrifuge at 12000 rpm;
(4) after the centrifugation is finished, transferring the supernatant into a new EP tube, determining the protein concentration by referring to a BCA method, adding a loading buffer to adjust the protein concentration, and boiling for 5min at 95 ℃ to obtain a protein sample;
(5) preparing 12% of separation glue and 5% of concentrated glue, mounting an electrophoresis frame, and carrying out sample application;
(6) after sample application, 80V till bromophenol blue reaches a boundary, and then the voltage is adjusted to be 120V till the bromophenol blue completely runs out;
(7) after the electric transfer clamp is installed, adjusting the parameters of the electric transfer instrument to be constant current of 250mA, electrically transferring for 90min, and after the electric transfer is finished, taking out the membrane, placing the membrane in 5% of skimmed milk powder, and sealing for 1h at room temperature;
(8) configuring a cleared-Caspase 3, a cleared-Caspase 9 and a beta-actin primary antibody to perform primary antibody incubation, and incubating overnight at 4 ℃;
(9) after the incubation is finished, recovering the primary antibody, washing the membrane by using TBST, incubating the corresponding secondary antibody after membrane washing, placing the secondary antibody on a shaking table, and incubating for 1 h;
(10) after the incubation was completed, the secondary antibody was removed, and the membrane was washed with TBST, and then developed with a developer.
The results of the examples are shown in FIG. 1, and it can be seen that the expression levels of clear-Caspase 3 and clear-Caspase 9 in example 5 are significantly lower compared to example 1, indicating that the addition of 200. mu.M of the peptide of the anus frog significantly reduces the apoptosis of the adipose-derived stem cells caused by long-term cryopreservation.
Example 3
Detection of fat-forming conversion ability
(1) After the cells of example 1 and example 5 were frozen for 12 months, the cells were thawed in a 37 ℃ water bath;
(2) after recovery, the cells are inoculated in a 6-hole cell culture plate, when the cell density reaches 95%, the cell is replaced by an alpha-MEM complete culture medium containing 5 mu g/ml of insulin, 0.25mM IBMX, 0.25 mu M dexamethasone and 50 mu M indomethacin, and after 3 days of cell culture, the cell is replaced by an alpha-MEM complete culture medium containing 5 mu g/ml of insulin;
(3) after 8 days of adipogenic induction, the culture medium was removed, rinsed 2 times with PBS and 500. mu.l of 4% formaldehyde was added to fix the cells for 15 min;
(4) after fixation is finished, removing the fixation solution, adding 500ul of 60% isopropanol solution for treating for 1min after PBS rinsing, removing, adding oil red O staining solution, and staining for 10 min;
(5) after removal, rinsing with deionized water, and then taking a picture under a microscope;
(6) thereafter, 500ul of 100% isopropanol was added to each well, incubated for 20min, and the extracted oil red O was transferred to a new EP tube and detected at 540nm OD using a microplate reader.
The results obtained are shown in FIG. 2, in which the OD values of example 1 were 0.776. + -. 0.055 and example 5 were 1.227. + -. 0.052, indicating that the frozen cells of example 5 had better adipogenic transformation ability. The results show that the addition of 200 mu M of the peptide of the rana amurensis can ensure that the human adipose-derived mesenchymal stem cells maintain better adipogenic transformation capacity during long-term cryopreservation.
Claims (2)
1. The adipose-derived stem cell cryopreservation liquid is characterized by comprising the following components: 40% DMEM/F12, 50% FBS, 10% DMSO, 100-250 mu M peptide of the said anus fold amino acid sequence Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-Val-Gly-Leu-Met-NH 2.
2. The cryopreservation liquid of claim 1, wherein the cryopreservation liquid comprises the following components: 40% DMEM/F12, 50% FBS, 10% DMSO, 200 μ M peptide of Rana temporaria.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102550542A (en) * | 2011-08-09 | 2012-07-11 | 臻景生物技术（上海）有限公司 | Establishment for serum-free freezing medium and adipose-derived stem cell library of adipose-derived stem cells |
| CN107156111A (en) * | 2017-06-30 | 2017-09-15 | 陈印平 | A kind of candidate stem cell cell cryopreservation agent |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102550542A (en) * | 2011-08-09 | 2012-07-11 | 臻景生物技术（上海）有限公司 | Establishment for serum-free freezing medium and adipose-derived stem cell library of adipose-derived stem cells |
| CN107156111A (en) * | 2017-06-30 | 2017-09-15 | 陈印平 | A kind of candidate stem cell cell cryopreservation agent |
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