CN112655700A - Application of frozen stock solution in gallbladder stem cells and recovery method of gallbladder stem cells - Google Patents
Application of frozen stock solution in gallbladder stem cells and recovery method of gallbladder stem cells Download PDFInfo
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Abstract
The invention provides an application of a freezing medium in gallbladder stem cells and a method for recovering the gallbladder stem cells, wherein the freezing medium comprises: 3-6 v/v% of dimethyl sulfoxide, 8-12 w/v% of human serum albumin, DMEM/F12 culture medium, N-acetylcysteine and an inhibitor Y-27632; the resuscitation system comprises a resuscitation method and a resuscitation fluid, wherein the resuscitation fluid comprises: human serum albumin and DMEM/F12 medium; after the cells in the cell freezing solution and the cell thawing system are frozen and thawed, the survival rate and the thawing rate still reach over 90 percent, and are superior to the traditional freezing solution; in addition, the frozen stock solution does not contain fetal bovine serum components, so that anaphylactic reaction caused by animal-derived protein and pollution intervention of related animal viruses are completely avoided; the content of dimethyl sulfoxide is reduced, and the influence of the toxicity of the frozen stock solution on the survival rate of stem cells is reduced; DMEM/F12 medium and human serum albumin contain rich nutrient elements, so that the survival rate of cells is guaranteed.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to application of a freezing solution in gallbladder stem cells and a method for recovering the gallbladder stem cells.
Background
Stem cells are cells having self-renewal ability and multi-differentiation potential, and by these properties, stem cells play an important role in development, growth, injury repair, immune regulation, and the like of the body. Meanwhile, the stem cells have wide application prospects in the fields of clinical treatment and regenerative medicine.
The gallbladder stem cells are derived from intrahepatic bile duct, bile duct or gallbladder epithelial cells, have strong proliferation capacity under proper culture conditions in vitro, and can be stably amplified. Under specific culture conditions, the gallbladder stem cells can specifically differentiate hepatocyte-like cells and bile duct cells in vitro. These all provide important bases for the application of the gall bladder stem cells in the treatment of liver and biliary duct system diseases.
The gallbladder stem cells are limited in separation and acquisition quantity, large in clinical application amount, and therefore need to be amplified in vitro in large quantity, and meanwhile, in order to ensure the quality of the cells and the stability of the cells during application, the cells need to be frozen at a low temperature according to the cell passage times in the amplification process. At present, the cell cryopreservation technology is generally a liquid nitrogen cryopreservation method, and cells are slowly frozen by adding a proper amount of protective agent, such as most commonly used dimethyl sulfoxide. If the protective agent is not added, the cells are directly frozen, and water inside and outside the cells can quickly form ice crystals, so that a series of changes can occur in the cells, such as mechanical damage, electrolyte concentration rise, osmotic pressure change, dehydration, pH value change, protein denaturation and the like, and cell death is caused. And the high-concentration dimethyl sulfoxide has certain biological toxicity, can reduce the cell survival rate and induce the stem cell differentiation. The other most commonly used cell protection nutrient component of fetal calf serum contains animal protein components, possibly remains in cells after cryopreservation and recovery, is easy to cause human body adverse immune response when applied to clinical treatment, and simultaneously, the fetal calf serum carries animal viruses and has the risk of causing human infection. In addition, the gallbladder stem cells have extremely high requirements on the storage environment, the traditional serum-free freezing solution and recovery system can cause the stem cells to die in a large proportion due to freezing, the requirements of clinical large-scale application are seriously influenced, and no better serum-free freezing solution and recovery system which can meet the freezing requirement of the stem cells with high survival rate exist in the field of the stem cells at present. Therefore, the freezing storage solution and the recovery system of the gallbladder stem cells with high efficiency and high survival rate need to be solved urgently.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the application of the frozen stock solution in the gallbladder stem cells.
The second purpose of the invention is to provide a gall bladder stem cell resuscitation solution.
The third purpose of the invention is to provide a method for recovering gallbladder stem cells.
The fourth purpose of the invention is to provide an application of the freezing medium in the freezing recovery of the gall bladder stem cells.
In order to achieve the above primary object, the solution of the present invention is:
application of frozen stock solution in gallbladder stem cells is provided.
Preferably, the cryopreservation solution comprises the following components: 3-6 v/v% of dimethyl sulfoxide, 8-12 w/v% of human serum albumin, 40-50 v/v% of DMEM/F12 culture medium, N-acetylcysteine with the concentration of 0.5-2mmol/L and inhibitor Y-27632 with the concentration of 5-15 mu mol/L.
In order to achieve the second objective, the solution of the invention is:
a gallbladder stem cell resuscitation solution comprises the following components: 3-5 w/v% human serum albumin and 75-85 v/v% DMEM/F12 medium.
Preferably, the concentration of human serum albumin is 4 w/v% and the concentration of DMEM/F12 medium is 80 v/v%.
In order to achieve the third object, the solution of the invention is:
a method for recovering gallbladder stem cells utilizes the gallbladder stem cell recovery liquid to recover the gallbladder stem cells.
Preferably, the resuscitation method comprises the steps of: preparing normal-temperature gall bladder stem cell resuscitating liquid with the same volume as the frozen cell suspension in a 15mL centrifuge tube in advance, transferring the frozen cell suspension which is quickly dissolved at 37 ℃ into the gall bladder stem cell resuscitating liquid, adding dropwise and uniformly mixing by shaking during transfer, continuously and slowly adding DMEM/F12 culture medium with 9 times of the volume of the frozen cell suspension, uniformly mixing by blowing, performing centrifugal collection and performing subsequent inoculation.
Further preferably, the resuscitation method comprises:
(1) and preparing the frozen stock solution: the above-described frozen stock solution was prepared.
(2) And preparing a frozen cell suspension: after in vitro culture of the gallbladder stem cells, centrifuging, mixing the collected gallbladder stem cells with a frozen stock solution for resuspension to obtain a cell suspension, and placing the cell suspension in a sterile frozen stock tube;
freezing: cooling the freezing tube to-80 ℃ for 24-72h, and then transferring to liquid nitrogen for freezing and storing;
cell recovery: and taking out the cryopreservation tube, placing the tube in a water bath at 37 ℃ for rapid dissolution, and shaking until the cryopreserved cell suspension is completely melted.
To achieve the fourth object, the solution of the present invention is:
an application of frozen stock solution in freezing storage and recovery of gallbladder stem cells is disclosed.
Due to the adoption of the scheme, the invention has the beneficial effects that:
the gall bladder stem cell cryopreservation liquid and the cryopreservation resuscitation system (the cryopreservation resuscitation liquid and the cryopreservation resuscitation method) do not contain fetal bovine serum components, so that anaphylactic reaction caused by animal-derived proteins and pollution intervention of related animal viruses are completely avoided; the content of dimethyl sulfoxide is reduced, and the influence of the toxicity of the frozen stock solution on the survival rate of cells is reduced; in addition, the DMEM/F12 culture medium and the human serum albumin contain rich nutrient elements, so that a stable nutrient environment is provided for cells, and the survival rate of the cells is ensured; by utilizing the recovery system provided by the invention, the recovery rate and the survival rate of cells can reach more than 90 percent, and the recovery system is superior to the traditional freezing solution and recovery system.
Drawings
FIG. 1 is a graph showing the survival rate of resuscitated cells in example 1 of the present invention.
FIG. 2 is a graph showing the survival rate of resuscitated cells in example 2 of the present invention.
FIG. 3 is a graph showing the survival rate of resuscitated cells in example 3 of the present invention.
FIG. 4 is a graph showing the recovery rate of the resuscitated cells in example 3 of the present invention.
Detailed Description
The invention provides application of a freezing medium in gallbladder stem cells and a recovery method of the gallbladder stem cells.
The cryopreservation solution can be applied to gallbladder stem cells.
Wherein the frozen stock solution comprises the following components: the concentration of dimethyl sulfoxide (DMSO) is preferably 3-6 v/v%, more preferably 4-5 v/v%, and still more preferably 5 v/v%; the concentration of human serum albumin is preferably 8-12 w/v%, more preferably 9-11 w/v%, and still more preferably 10 w/v%; the concentration of DMEM/F12 medium is preferably 40-50 v/v%, more preferably 45 v/v%; the concentration of the N-acetylcysteine is 0.5-2mmol/L, preferably 1 mmol/L; the concentration of inhibitor Y-27632 is 5-15. mu. mol/L, preferably 10. mu. mol/L.
The recovery system comprises a recovery method of the gall bladder stem cells and a gall bladder stem cell recovery solution.
The gallbladder stem cell recovery liquid comprises the following components: the concentration of human serum albumin is preferably 3-5 w/v%, more preferably 4 w/v%; the concentration of DMEM/F12 medium is preferably 75-85 v/v%, more preferably 80 v/v%.
The method for recovering the gall bladder stem cells comprises the following steps: and (3) recovering the gallbladder stem cells by using the gallbladder stem cell recovery liquid.
Specifically, the resuscitation method comprises the following steps:
preparing normal-temperature gall bladder stem cell resuscitating liquid with the same volume as the frozen cell suspension in a 15mL centrifuge tube in advance, rapidly dissolving cells taken out of liquid nitrogen in water at 37 ℃, transferring the dissolved frozen cell suspension into the gall bladder stem cell resuscitating liquid, shaking and uniformly mixing while dropwise adding during transfer, then continuously and slowly adding DMEM/F12 culture medium with 9 times of the volume of the frozen cell suspension, slightly blowing and uniformly mixing, and performing centrifugal collection and subsequent inoculation steps.
Further specifically, the resuscitation method comprises:
(1) and preparing the frozen stock solution: the above-described frozen stock solution was prepared.
(2) And preparing a frozen cell suspension: after in vitro culture of the gallbladder stem cells, centrifuging, mixing the collected gallbladder stem cells with a frozen stock solution for resuspension to obtain a cell suspension, and placing the cell suspension in a sterile frozen stock tube;
freezing: cooling the freezing tube to-80 ℃ for 24-72h, and then transferring to liquid nitrogen for freezing and storing;
cell recovery: and taking out the cryopreservation tube, and placing the tube in a water bath at 37 ℃ for rapid dissolution to obtain the cryopreservation cell suspension.
The cryopreservation liquid can be applied to cryopreservation and resuscitation of the gallbladder stem cells.
The present invention will be further described with reference to the following examples.
Example 1:
the gallbladder stem cell recovery liquid comprises the following components: 3-5 w/v% human serum albumin and 75-85 v/v% DMEM/F12 medium.
The frozen stock solution consists of the components of the conventional serum-free stem cell frozen stock solution, and specifically comprises the following components: 10 v/v% Dimethylsulfoxide (DMSO), 3 v/v% human serum albumin and 75 v/v% DMEM/F12 medium.
The resuscitation method of the embodiment comprises the following steps:
(1) and after the primary separated human gallbladder stem cells are cultured in vitro for 2-3 generations, removing impurity cells and enriching high-purity gallbladder stem cells.
(2) And digesting the gallbladder stem cells in vitro by using Gibco TrypLE digestive enzyme, collecting and suspending in DMEM/F12, centrifuging at 300 Xg for 5min, and discarding the supernatant. Resuspending cells with cell freezing medium at 4 deg.C, collecting 20 μ L cell suspension, mixing according to cell suspension (v) 0.08% trypan blue (v) 1:1, and calculating cell viability and quantity; adjusting the cell density to 1 × 106cells/mL, the cell suspension is subpackaged into freezing storage tubes, 1.0 mL/tube; and (6) marking.
(3) And putting the freezing tube filled with the cell suspension into a gradient freezing box, putting the gradient freezing box into a refrigerator at minus 80 ℃, and transferring the gradient freezing box into liquid nitrogen after 24 hours.
(4) And the cells are thawed after being frozen in liquid nitrogen for 7 days, and the method comprises the following steps: the cells were removed from the liquid nitrogen, rapidly lysed in water at 37 ℃ and then resuscitated into three resuscitation regimes:
a first group: directly and quickly transferring the frozen cell suspension into a DMEM/F12 culture medium buffer solution with the volume 10 times that of the frozen cell suspension, and carrying out centrifugal collection;
second group: slowly dropwise adding the frozen cell suspension into DMEM/F12 culture medium buffer solution with the volume 10 times that of the frozen cell suspension, uniformly mixing the frozen cell suspension and the DMEM/F12 culture medium buffer solution while dropwise adding, and performing centrifugal collection;
third group: transferring the frozen cell suspension into a 15mL centrifuge tube containing a gall bladder stem cell resuscitation solution with the same volume as the frozen cell suspension, uniformly mixing the frozen cell suspension and the 15mL centrifuge tube while dropping the cell suspension, continuously adding a DMEM/F12 culture medium with the volume 9 times that of the frozen cell suspension, uniformly mixing the DMEM/F12 culture medium and performing centrifugal collection;
centrifuging for 5min at 300 Xg, discarding the supernatant, resuspending and mixing with DMEM/F12 culture medium with the volume 3 times of that of the frozen cell suspension, centrifuging for 5min at 300 Xg, discarding the supernatant, resuspending and mixing with DMEM/F12 culture medium with the same volume of the frozen cell suspension, taking 20 mu L of cell suspension, mixing according to the cell suspension (v): 0.08% trypan blue (v): 1, and calculating the cell viability and the number, wherein the results are shown in figure 1.
As can be seen from FIG. 1, the gallbladder stem cells are recovered after being frozen in the conventional serum-free stem cell freezing medium, the cell survival rate is extremely low, and the requirement of the gallbladder stem cells on the culture environment is high. By using the resuscitation system of the embodiment, the resuscitation rate of the gallbladder stem cells can be still greatly improved and is obviously higher than that of the conventional resuscitation method.
Example 2: comparison of different frozen stock solution components of Stem cells
The recovery survival rates of the gallbladder stem cells after the cryopreservation of the following different freezing solutions are compared, namely the embodiment compares the influence of different added components on the cryopreservation efficiency and is prepared by adding other components on the basis of the traditional common freezing solution.
A first group: dimethylsulfoxide (10 v/v%), human serum albumin (3 w/v%) and DMEM/F12 medium (75 v/v%).
Second group: dimethyl sulfoxide (10 v/v%), human serum albumin (3 w/v%), DMEM/F12 medium (75 v/v%) and N-acetylcysteine (1 mmol/L).
Third group: dimethyl sulfoxide (10 v/v%), human serum albumin (3 w/v%), DMEM/F12 medium (75 v/v%) and Y-27632 (10. mu. mol/L).
And a fourth group: dimethyl sulfoxide (10 v/v%), human serum albumin (3 w/v%), DMEM/F12 medium (75 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
The resuscitation method of the embodiment comprises the following steps:
(1) and after the primary separated human gallbladder stem cells are cultured in vitro for 2-3 generations, removing impurity cells and enriching high-purity gallbladder stem cells.
(2) And digesting the gallbladder stem cells in vitro by using Gibco TrypLE digestive enzyme, collecting and suspending the gallbladder stem cells in DMEM/F12, averagely dividing the gallbladder stem cells into four groups of cell suspensions, centrifuging the cell suspensions at 300 Xg for 5min, and then discarding the supernatant. Resuspending cells with four groups of cell freezing solutions with different components at a temperature of more than 4 ℃, respectively taking 20 mu L of cell suspension, and uniformly mixing the cell suspension according to the ratio of 0.08% trypan blue (v) to 1:1 to calculate the cell viability and the number; adjusting the cell density to 1 × 106cells/mL, the cell suspension is subpackaged into freezing storage tubes, 1.0 mL/tube; and (6) marking.
(3) And putting the freezing tube filled with the cell suspension into a gradient freezing box, putting the gradient freezing box into a refrigerator at minus 80 ℃, and transferring the gradient freezing box into liquid nitrogen after 72 hours.
(4) And the cells are thawed after being frozen in liquid nitrogen for 7 days, and the method comprises the following steps: taking out cells from liquid nitrogen, quickly dissolving the cells in water at 37 ℃, transferring the cell suspension into a 15mL centrifuge tube containing resuscitating fluid with the same volume as that of the frozen cell suspension, uniformly mixing the cells while dropping the cells during transfer, gently blowing the cells by using a pipette after the transfer is finished, continuously adding DMEM/F12 culture medium with 9 times of the volume of the frozen cell suspension, fully mixing the cells, centrifuging the cells at 300 Xg for 5min, re-suspending and uniformly mixing the cells by using DMEM/F12 culture medium with 3 times of the volume of the frozen cell suspension after supernatant is removed, centrifuging the cells at 300 Xg for 5min, re-suspending and uniformly mixing the cells by using DMEM/F12 culture medium with the same volume of the frozen cell suspension after supernatant is removed, taking 20 mu L of the cell suspension, uniformly mixing the cell suspension (v) and 0.08% trypan (v) ratio of 1:1, and calculating the cell activity and the number.
The results show that the freezing protection effect of the gallbladder stem cells is improved to a certain extent after the N-acetylcysteine is added separately to the second group, the freezing protection effect of the gallbladder stem cells is also improved to a certain extent after the micromolecular inhibitor Y-27632 is added separately to the third group, and the freezing protection effect is further improved by the combined protection effect of the N-acetylcysteine and the micromolecular inhibitor Y-27632 which are added simultaneously to the fourth group.
Example 3: comparison of recovery activity rates of different freezing solutions
And comparing the recovery survival rates of the gallbladder stem cells after the cryopreservation of the cryopreservation solutions with different concentrations of the following components.
A first group: DMEM/F12 medium (15 v/v%), human serum albumin (15 w/v%), DMSO (10 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
Second group: DMEM/F12 medium (40 v/v%), human serum albumin (10 w/v%), DMSO (10 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
Third group: DMEM/F12 medium (60 v/v%), human serum albumin (6 w/v%), DMSO (10 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
And a fourth group: DMEM/F12 medium (75 v/v%), human serum albumin (3 w/v%), DMSO (10 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
And a fifth group: DMEM/F12 medium (17.5 v/v%), human serum albumin (15 w/v%), DMSO (7.5 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
A sixth group: DMEM/F12 medium (42.5 v/v%), human serum albumin (10 w/v%), DMSO (7.5 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
A seventh group: DMEM/F12 medium (62.5 v/v%), human serum albumin (6 w/v%), DMSO (7.5 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
And an eighth group: DMEM/F12 medium (77.5 v/v%), human serum albumin (3 w/v%), DMSO (7.5 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
Ninth group: DMEM/F12 medium (20 v/v%), human serum albumin (15 w/v%), DMSO (5 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
The tenth group: DMEM/F12 medium (45 v/v%), human serum albumin (10 w/v%), DMSO (5 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
Eleventh group: DMEM/F12 medium (65 v/v%), human serum albumin (6 w/v%), DMSO (5 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
A twelfth group: DMEM/F12 medium (80 v/v%), human serum albumin (3 w/v%), DMSO (5 v/v%), N-acetylcysteine (1mmol/L) and Y-27632 (10. mu. mol/L).
The resuscitation method of the embodiment comprises the following steps:
(1) and after the primary separated human gallbladder stem cells are cultured in vitro for 2-3 generations, removing impurity cells and enriching high-purity gallbladder stem cells.
(2) And digesting the gallbladder stem cells in vitro by using Gibco TrypLE digestive enzyme, collecting and suspending the gallbladder stem cells in DMEM/F12, averagely dividing the gallbladder stem cells into four groups of cell suspensions, centrifuging the cell suspensions at 300 Xg for 5min, and then discarding the supernatant. Resuspending cells with four groups of cell freezing solutions with different components at a temperature of more than 4 ℃, respectively taking 20 mu L of cell suspension, and uniformly mixing the cell suspension according to the ratio of 0.08% trypan blue (v) to 1:1 to calculate the cell viability and the number; adjusting the cell density to 1 × 106cells/mL, the cell suspension is subpackaged into freezing storage tubes, 1.0 mL/tube; and (6) marking.
(3) And putting the freezing tube filled with the cell suspension into a gradient freezing box, putting the gradient freezing box into a refrigerator at the temperature of-80 ℃, and transferring the gradient freezing box into liquid nitrogen after 36 hours.
(4) And the cells are thawed after being frozen in liquid nitrogen for 7 days, and the method comprises the following steps: taking out cells from liquid nitrogen, quickly dissolving the cells in water at 37 ℃, transferring the cell suspension into a 15mL centrifuge tube containing resuscitating fluid with the same volume as that of the frozen cell suspension, uniformly mixing the cells while dropping the cells during transfer, gently blowing the cells by using a pipette after the transfer is finished, continuously adding DMEM/F12 culture medium with 9 times of the volume of the frozen cell suspension, fully mixing the cells, centrifuging the cells at 300 Xg for 5min, re-suspending and uniformly mixing the cells by using DMEM/F12 culture medium with 3 times of the volume of the frozen cell suspension after supernatant is removed, centrifuging the cells at 300 Xg for 5min, re-suspending and uniformly mixing the cells by using DMEM/F12 culture medium with the same volume of the frozen cell suspension after supernatant is removed, taking 20 mu L of the cell suspension, uniformly mixing the cell suspension (v) and 0.08% trypan (v) ratio of 1:1, and calculating the cell activity and the number, wherein the results.
The results show that the survival rate of the cells preserved by the freezing medium of the tenth group after recovery reaches 95.35 +/-1.25 percent, and the cell recovery rate reaches 99.05 percent, so that the concentration of each component of the freezing medium of the group is the optimal concentration of the freezing medium of the gallbladder stem cells.
As described above, the cryopreservation solution of example 1, which uses a conventional stem cell serum-free cryopreservation solution, can demonstrate the superiority of the resuscitation system, but the overall resuscitation rate is still not high. The two components, namely N-acetylcysteine and the inhibitor Y-27632, are added into the frozen stock solution in the example 2, and the frozen stock recovery survival rate is improved again after the two components are added simultaneously. Example 3 the frozen stock solution was adjusted for its component content, i.e. for its concentration, DMSO, human serum albumin and DMEM/F12, to obtain a tenth group with very high resuscitation activity and resuscitation efficiency at the frozen stock solution component content.
The cell freezing solution provided by the invention does not contain fetal bovine serum components, so that anaphylactic reaction and pollution introduction of animal viruses are completely avoided; meanwhile, the content of DMSO (dimethyl sulfoxide) is reduced, and the influence of the toxicity of the frozen stock solution on cells is further reduced. In addition, the cell recovery system provided by the invention improves the survival rate of cell recovery after cryopreservation by an optimized recovery method and recovery liquid. Experiments prove that the cryopreservation liquid and the resuscitation system provided by the invention are superior to the traditional cryopreservation liquid and resuscitation system. Therefore, the stem cell recovery rate after long-time cryopreservation by using the stem cell cryopreservation liquid and the recovery system provided by the invention is higher.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.
Claims (8)
1. Application of frozen stock solution in gallbladder stem cells is provided.
2. Use according to claim 1, characterized in that: the freezing solution comprises the following components: 3-6 v/v% of dimethyl sulfoxide, 8-12 w/v% of human serum albumin, 40-50 v/v% of DMEM/F12 culture medium, N-acetylcysteine with the concentration of 0.5-2mmol/L and inhibitor Y-27632 with the concentration of 5-15 mu mol/L.
3. A gall bladder stem cell resuscitation fluid is characterized in that: the paint comprises the following components: 3-5 w/v% human serum albumin and 75-85 v/v% DMEM/F12 medium.
4. The gallbladder stem cell resuscitation fluid of claim 3, wherein: the concentration of the human serum albumin is 4 w/v%, and the concentration of the DMEM/F12 culture medium is 80 v/v%.
5. A method for recovering gallbladder stem cells, wherein the gallbladder stem cells are recovered by using the gallbladder stem cell recovering solution of claim 3;
the gallbladder stem cell is the gallbladder stem cell of claim 1.
6. The resuscitation method according to claim 5, wherein: which comprises the following steps:
dissolving the frozen cell suspension at 37 ℃, then mixing the dissolved cell suspension with the gall bladder stem cell recovery solution of claim 3 drop by drop, continuing to add DMEM/F12 culture medium, and centrifugally collecting.
7. The resuscitation method according to claim 6, wherein: it includes:
(1) and preparing the frozen stock solution: preparing the frozen stock solution of claim 1.
(2) And preparing a frozen cell suspension: after in vitro culture of the gallbladder stem cells, centrifuging, mixing the collected gallbladder stem cells with the frozen stock solution for resuspension to obtain a cell suspension, and placing the cell suspension in a freezing tube;
freezing: cooling the freezing tube to-80 ℃ for 24-72h, and then transferring to liquid nitrogen for freezing and storing;
cell recovery: taking out the frozen tube, and placing the tube in a water bath at 37 ℃ to dissolve the tube into frozen cell suspension.
8. An application of frozen stock solution in freezing storage and recovery of gallbladder stem cells is disclosed.
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| CN114946836A (en) * | 2022-05-25 | 2022-08-30 | 成都诺医德医学检验实验室有限公司 | Micro-tissue serum-free freezing medium and application thereof |
| CN114946836B (en) * | 2022-05-25 | 2024-06-04 | 成都诺医德医学检验实验室有限公司 | Micro-tissue serum-free frozen stock solution and application thereof |
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