CN111685103A - Cryopreservation method for umbilical cord blood hematopoietic stem cells - Google Patents
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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
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- A—HUMAN NECESSITIES
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention provides a cryopreservation method of umbilical cord blood hematopoietic stem cells, which comprises the following steps: uniformly mixing umbilical cord blood hematopoietic stem cells, dextran and DMSO according to the volume ratio of 8:1:1 to form a mixed solution; filling the mixed solution into a freezing storage bag; the freezing bag is kept at a first temperature T1 for a first time period T1; the freezing bag is placed in the environment with the second temperature T2 for a second time period T2; the freezing bag is placed in an environment with a third temperature T3 for a third time period T3; the frozen storage bag is stored in an environment of a fourth temperature T4. The invention has the beneficial effects that: the method for cryopreserving the umbilical cord blood hematopoietic stem cells at the fixed temperature has the characteristics of simple operation, safety, reliability, low cost and high cell resuscitation and survival rate, and is very suitable for large-scale umbilical cord blood hematopoietic stem cell cryopreservation.
Description
Technical Field
The invention relates to the technical field of cell cryopreservation, in particular to a method for cryopreserving umbilical cord blood hematopoietic stem cells.
Background
At present, stem cell therapy has been extensively studied and popularized at home and abroad, and hematopoietic stem cells are the only clinically applicable stem cell resource in the present stem cell family. The transplantation of cord blood hematopoietic stem cells can treat hundreds of diseases of blood system, immune system, genetic metabolism and the like. With the continuous development of science and technology, the application prospect of the cord blood hematopoietic stem cells is wider.
Scientists have long demonstrated that cells can be stored in low temperature environments for long periods of time. However, direct exposure of cells to low temperature conditions can cause damage to the cells by ice crystals or solutes, which are irreversible and can affect the viability of the cells after recovery. Further research shows that the ice crystal or solute damage can be obviously reduced by cooling the cells added with the cryoprotectant at a certain rate, and the recovery survival rate of the cells is improved. At present, isopropanol program cooling boxes or program cooling instruments are commonly used for cooling cells, and then the cells are transferred to a gas-phase liquid nitrogen storage tank for storage. However, no program cooling box aiming at the freezing storage bags with the specification of 25ml/50ml is available in the market at present, so that the program cooling can be only carried out on the freezing storage bags with the specification of 25ml/50ml by adopting a program cooling instrument.
The prior art is not enough:
the program cooling instrument is used for program cooling, the equipment price is high, liquid nitrogen is used as a cold source of the program cooling instrument, and due to the special production and storage conditions of the liquid nitrogen, the liquid nitrogen cost is high, so that the cell cost for cooling by using the program cooling instrument is high.
The excess nitrogen generated by the vaporization of liquid nitrogen under normal pressure can reduce the oxygen partial pressure in the air, and can cause oxygen deficiency asphyxiation of people under extreme conditions. The liquid nitrogen temperature is-196 ℃, which is easy to cause the risk of accidental contact and frostbite of personnel.
The program cooling instrument is a high-precision instrument, the operation is relatively complex, and operators can be trained for a long time before the operators are on duty.
The programmed cooling of cells by using the programmed cooling instrument is influenced by the volume of the programmed cooling instrument and the supply of liquid nitrogen, and the programmed cooling quantity of single cells is limited, so that the large-scale production of the umbilical cord blood hematopoietic stem cells at present cannot be met.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a cryopreservation method which has convenient operation, safety, reliability, low cost and high cell resuscitation survival rate.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for cryopreserving umbilical cord blood hematopoietic stem cells, comprising the following steps:
s1, mixing the cord blood hematopoietic stem cells, dextran and DMSO uniformly according to the volume ratio of 8:1:1 to form a mixed solution;
s2, filling the mixed solution into a freezing storage bag;
s3, the frozen bag is placed in an environment with a first temperature T1 and kept for a first time length T1, wherein the temperature is higher than 3 ℃ and lower than T1 and lower than 5 ℃, and the temperature is higher than 25min and lower than T1 and lower than 35 min;
s4, keeping the freezing storage bag at a second temperature T2 for a second time period T2, wherein T2 is more than-25 ℃ and less than-15 ℃, and T2 is more than 110min and less than 130 min;
s5, keeping the freezing bag at a third temperature T3 for a third time period T3, wherein T3 is more than 85 ℃ below zero and less than 75 ℃ below zero, and T3 is more than 11H and less than 13H;
s6, storing the frozen bag in an environment with a fourth temperature T4, wherein the T4 is less than or equal to-150 ℃.
Further, in step S1, the cells are concentrated at 2 × 107The umbilical cord blood hematopoietic stem cells, 10% dextran and 10% DMSO are uniformly mixed according to the volume ratio of 8:1:1 to form a mixed solution.
Further, the first temperature T1 is 4 ℃, and the first time length T1 is 30 min;
-20 ℃ for the second temperature T2, 2H for the second time period T2;
the third temperature T3 ═ 80 ℃, and the third time period T3 ═ 12H.
Further, in step S2, the capacity of the cryopreservation bag is L, wherein L is more than or equal to 25ml and less than or equal to 50 ml.
Further, in the step of maintaining the cryopreservation bag at the environment of the second temperature for the second time period, the transfer temperature is not higher than T1;
during the step of maintaining the cryopreservation bag at the environment of the third temperature for the third period of time, the transfer temperature is not higher than T2;
in the step of storing the cryopreservation bag in an environment of a fourth temperature T4, the transfer temperature is not higher than T3.
Further, in step S1, the cord blood hematopoietic stem cells, dextran, and DMSO are mixed uniformly to form a mixed solution by blowing and beating the tip multiple times.
Further, in the step of keeping the freezing and storing bag at the first temperature for a first time, continuously preserving heat of the freezing and storing bag by adopting a first heat preservation box;
in the step of keeping the freezing bag at the second temperature for a second time, a second heat preservation box is adopted to continuously preserve heat of the freezing bag;
and in the step of keeping the freezing bag at the third temperature for the third time, continuously preserving the heat of the freezing bag by using a third heat preservation box.
Further, after the step S6, the method further comprises the step of resuscitating the cord blood stem cells in the frozen storage bag, specifically, resuscitating the frozen storage bag in a water bath at 37 ℃ for 2 minutes.
Further, after the step of putting the frozen bag in a water bath at 37 ℃ for 2 minutes for resuscitation, the method also comprises the step of determining the viability of the cord blood stem cells.
Further, in step S6, the frozen storage bag is stored in the environment of the fourth temperature T4, and the storage time T0 is more than or equal to 1M.
The invention has the beneficial effects that: the method for cryopreserving the umbilical cord blood hematopoietic stem cells at the fixed temperature has the characteristics of simple operation, safety, reliability, low cost and high cell resuscitation and survival rate, and is very suitable for large-scale umbilical cord blood hematopoietic stem cell cryopreservation.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the description of the invention relating to "first", "second", etc. is for descriptive purposes only and is not to be construed as indicating or implying any relative importance or implicit indication of the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
Example 1
A method for cryopreserving umbilical cord blood hematopoietic stem cells, comprising the following steps:
s1, the concentration of the cells is 2 x 107Uniformly mixing umbilical cord blood hematopoietic stem cells, 10% dextran and 10% DMSO according to the volume ratio of 8:1:1 to form a mixed solution;
s2, filling the mixed solution into a freezing storage bag, wherein the freezing storage bag is an aseptic freezing storage bag with the specification of 25ml/50 ml;
s3, keeping the freezing storage bag at a first temperature T1 for a first time period T1, and continuously preserving heat of the freezing storage bag by using a first heat preservation box, wherein the temperature T1 is 4 ℃, the temperature T1 is 30min, and the temperature of the first heat preservation box is 4 ℃ medical heat preservation box;
s4, keeping the freezing and storing bag in an environment with a second temperature T2 for a second time period T2, and continuously preserving heat of the freezing and storing bag by adopting a second heat preservation box, wherein the temperature T2 is-20 ℃, the temperature T2 is 120min, and the second heat preservation box is a low-temperature preservation box with the temperature of-20 ℃;
s5, keeping the freezing and storing bag in an environment with a third temperature T3 for a third time period T3, and continuously preserving heat of the freezing and storing bag by using a third heat preservation box, wherein the temperature T3 is-80 ℃, the temperature T3 is 12Hour, and the third heat preservation box is a deep low temperature storage box with the temperature of-80 ℃;
s6, storing the freezing bag in an environment with a fourth temperature T4, and storing the freezing bag by adopting a fourth heat preservation box, wherein the T4 is less than or equal to-150 ℃, and the fourth heat preservation box is a gas phase liquid nitrogen storage tank with the temperature of less than-150 ℃.
Experimental example 1
S1, the concentration of the cells is 2 x 107Uniformly mixing umbilical cord blood hematopoietic stem cells, 10% dextran and 10% DMSO according to the volume ratio of 8:1:1 to form a mixed solution;
s2, filling the mixed solution into a freezing storage bag;
s3, keeping the freezing bag at a first temperature T1 for a first time period T1, wherein T1 is 3 ℃ and T1 is 25 min;
s4, keeping the freezing bag at a second temperature T2 for a second time period T2, wherein T2 is-25 ℃, and T2 is 110 min;
s5, keeping the frozen bag at a third temperature T3 for a third time period T3, wherein T3 is-85 ℃, and T3 is 11.5 Hour;
s6, storing the frozen bag in an environment with a fourth temperature T4, wherein the T4 is less than or equal to-150 ℃.
Experimental example 2
S1, the concentration of the cells is 2 x 107Uniformly mixing umbilical cord blood hematopoietic stem cells, 10% dextran and 10% DMSO according to the volume ratio of 8:1:1 to form a mixed solution;
s2, filling the mixed solution into a freezing storage bag;
s3, keeping the freezing bag at a first temperature T1 for a first time period T1, wherein T1 is 5 ℃ and T1 is 35 min;
s4, keeping the freezing bag at a second temperature T2 for a second time period T2, wherein T2 is-15 ℃ and T2 is 130 min;
s5, keeping the frozen bag at a third temperature T3 for a third time period T3, wherein T3 is-75 ℃, and T3 is 12.5 Hour;
s6, storing the frozen bag in an environment with a fourth temperature T4, wherein the T4 is less than or equal to-150 ℃.
Example 1, experimental example 1 and experimental example 2 were cryopreserved and tested for resuscitation activity rates, which are shown in table 1:
| cryopreservation method | Rate of resuscitation |
| Example 1 | 90.03％ |
| Experimental example 1 | 87.83％ |
| Experimental example 2 | 88.75％ |
TABLE 1
The experimental results in the table show that the cell viability rates of the cryopreservation methods of the embodiment 1, the experiment 1 and the experiment 2 are all above 85%, and the cell cryopreservation viability rate requirements are met.
Example 2
The cells were concentrated at 2 x 107Uniformly mixing umbilical cord blood hematopoietic stem cells, 10% dextran and 10% DMSO according to the volume ratio of 8:1:1 to form a mixed solution;
filling the mixed solution into a freezing storage bag, wherein the freezing storage bag is an aseptic freezing storage bag with the specification of 25ml/50 ml;
keeping the freezing bag at a first temperature T1 for a first time period T1, wherein T1 is 4 ℃ and T1 is 30 min;
the frozen storage bags were stored at a third temperature T3, T3 ═ 80 ℃.
Example 3
The cells were concentrated at 2 x 107Per ml cord blood hematopoietic stem cell, 10% dextran and 10%Uniformly mixing DMSO according to the volume ratio of 8:1:1 to form a mixed solution;
filling the mixed solution into a freezing storage bag, wherein the freezing storage bag is an aseptic freezing storage bag with the specification of 25ml/50 ml;
keeping the freezing bag at a second temperature T2 for a second time period T2, wherein T2 is-20 ℃ and T2 is 120 min;
the frozen storage bags were stored at a third temperature T3, T3 ═ 80 ℃.
Experimental conditions 1
A25 ml sterile freezing bag is adopted, after the cells are preserved for one month, the freezing bag is placed in a water bath at 37 ℃ for 2 minutes by a conventional method, and then the survival rate is measured.
Experimental conditions 2
A sterile freezing bag with the specification of 50ml is adopted, after cells are preserved for one month, the freezing bag is placed in a water bath with the temperature of 37 ℃ for 2 minutes by a conventional method, and then the survival rate is measured.
Example 1, example 2, and example 3 were cryopreserved under test condition 1 and test condition 2, respectively, and the resuscitation rates were measured and shown in table 2:
TABLE 2
The experimental results in the table show that the cell survival rate of the cryopreservation method in the embodiment 1 is over 90 percent, and the cell cryopreservation survival rate requirement is met.
Comparative example 1
S101, setting the cell concentration to 2 x 107Uniformly mixing umbilical cord blood hematopoietic stem cells, 10% dextran and 10% DMSO according to the volume ratio of 8:1:1 to form a mixed solution;
s102, filling the mixed solution into a 25ml sterile freezing storage bag;
s103, placing 25ml of sterile freezing storage bags into a thermo 7453 programmed cooling instrument for cooling;
s104, placing the cooled 25ml frozen bag into a gas phase liquid nitrogen storage tank below 150 ℃ for storage.
The specific program design of the program cooling instrument in step S103 is as follows:
s1031, waiting until the temperature of the sample is close to 4 ℃;
s1032, cooling at the speed of 1 ℃/min until the temperature of the sample is minus 5.0 ℃;
s1033, cooling at a speed of 21 ℃/min until the temperature of the cavity is-54.0 ℃;
s1034, cooling at the speed of 17 ℃/min until the temperature of the cavity is minus 21.0 ℃;
s1035, cooling at the speed of 2 ℃/min until the temperature of the sample is minus 40.0 ℃;
s1036, operating at the speed of 10 ℃/min to cool until the temperature of the sample is minus 80.0 ℃;
s1037, the routine ends.
Comparative example 2
S201, setting the cell concentration to 2 x 107Uniformly mixing umbilical cord blood hematopoietic stem cells, 10% dextran and 10% DMSO according to the volume ratio of 8:1:1 to form a mixed solution;
s202, filling the mixed solution into a 25ml sterile freezing storage bag;
s203, putting the 25ml sterile freezing bag into a thermo 7453 programmed cooling instrument for cooling;
s104, putting the cooled 50ml frozen bag into a gas phase liquid nitrogen storage tank below 150 ℃ for storage.
The specific program design of the program cooling instrument in step S203 is as follows:
s2031, waiting until the temperature of the sample is close to 4 ℃;
s2032, cooling at a rate of 1 ℃/min until the temperature of the sample is-5.0 ℃;
s2033, cooling at a rate of 21 ℃/min until the temperature of the cavity is-54.0 ℃;
s2034, cooling at a speed of 17 ℃/min until the temperature of the cavity is-21.0 ℃;
s2035, cooling at a speed of 2 ℃/min until the temperature of the sample is minus 40.0 ℃;
s2036, cooling at the speed of 10 ℃/min until the temperature of the sample is minus 80.0 ℃;
s2037, the process ends.
After the cells were preserved for one month using the protocol of example 1 under experimental condition 1 and experimental condition 2, and the protocol of comparative example 1 and comparative example 2, the cryopreserved bag was placed in a water bath at 37 ℃ for 2 minutes by a conventional method, and then the survival rate was measured, and the obtained recovery survival rate was as shown in table 3:
TABLE 3
The results show that the cell viability rates of the schemes of the experimental condition 1, the experimental condition 2, the experimental condition 1, the comparative example 1 and the comparative example 2 are all over 90 percent, the requirement of the cell cryopreservation viability rate is met, and the schemes have no obvious difference.
In the process of adopting the schemes of the experimental condition 1, experimental condition 2, example 1, comparative example 1 and comparative example 2, the calculation of the cost of single cooling is performed on the power, the liquid nitrogen consumption and the like, and the cost calculation results shown in table 4 can be obtained:
| grouping | Cost/dollar |
| Experimental conditions 1 example 1 | 4.23 |
| Experimental conditions 2 example 1 | 4.23 |
| Comparative example 1 | 101.11 |
| Comparative example 2 | 101.11 |
TABLE 4
The result shows that the single cooling cost of the embodiment 1 of the experimental condition 1 is the same as that of the embodiment 1 of the experimental condition 2, and is far lower than that of the freezing cost of the embodiments of the comparative example 1 and the comparative example 2.
From the above description, the beneficial effects of the present invention are: the method for cryopreserving the umbilical cord blood hematopoietic stem cells at the fixed temperature has the characteristics of simple operation, safety, reliability, low cost and high cell resuscitation and survival rate, and is very suitable for large-scale umbilical cord blood hematopoietic stem cell cryopreservation.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A method for cryopreserving umbilical cord blood hematopoietic stem cells, comprising the following steps:
s1, mixing the cord blood hematopoietic stem cells, dextran and DMSO uniformly according to the volume ratio of 8:1:1 to form a mixed solution;
s2, filling the mixed solution into a freezing storage bag;
s3, the frozen bag is placed in an environment with a first temperature T1 and kept for a first time length T1, wherein the temperature is higher than 3 ℃ and lower than T1 and lower than 5 ℃, and the temperature is higher than 25min and lower than T1 and lower than 35 min;
s4, keeping the freezing storage bag at a second temperature T2 for a second time period T2, wherein T2 is more than-25 ℃ and less than-15 ℃, and T2 is more than 110min and less than 130 min;
s5, keeping the freezing bag at a third temperature T3 for a third time period T3, wherein T3 is more than 85 ℃ below zero and less than 75 ℃ below zero, and T3 is more than 11H and less than 13H;
s6, storing the frozen bag in an environment with a fourth temperature T4, wherein the T4 is less than or equal to-150 ℃.
2. The cryopreservation method of cord blood hematopoietic stem cells according to claim 1, wherein: in step S1, the cells are concentrated at 2 x 107The umbilical cord blood hematopoietic stem cells, 10% dextran and 10% DMSO are uniformly mixed according to the volume ratio of 8:1:1 to form a mixed solution.
3. The cryopreservation method of cord blood hematopoietic stem cells according to claim 2, wherein:
the first temperature T1 ═ 4 ℃, the first time length T1 ═ 30 min;
-20 ℃ for the second temperature T2, 2H for the second time period T2;
the third temperature T3 ═ 80 ℃, and the third time period T3 ═ 12H.
4. The method for cryopreserving umbilical cord blood hematopoietic stem cells according to claim 3, wherein: in step S2, the volume of the frozen bag is L, wherein L is more than or equal to 25ml and less than or equal to 50 ml.
5. The method for cryopreserving umbilical cord blood hematopoietic stem cells according to claim 4, wherein:
during the step of maintaining the cryopreservation bag in the environment at the second temperature for the second time period, the transfer temperature is not higher than T1;
during the step of maintaining the cryopreservation bag at the environment of the third temperature for the third period of time, the transfer temperature is not higher than T2;
in the step of storing the cryopreservation bag in an environment of a fourth temperature, the transfer temperature is not higher than T3.
6. The method for cryopreserving umbilical cord blood hematopoietic stem cells according to claim 5, wherein: in step S1, the cord blood hematopoietic stem cells, dextran, and DMSO are mixed uniformly to form a mixed solution by blowing and beating the tip several times.
7. The method for cryopreserving umbilical cord blood hematopoietic stem cells according to claim 6, wherein: in the step of keeping the freezing and storing bag at the first temperature for a first time, adopting a first heat preservation box to carry out continuous heat preservation on the freezing and storing bag;
in the step of keeping the freezing bag at the second temperature for a second time, a second heat preservation box is adopted to continuously preserve heat of the freezing bag;
and in the step of keeping the freezing bag at the third temperature for the third time, continuously preserving the heat of the freezing bag by using a third heat preservation box.
8. The method for cryopreserving umbilical cord blood hematopoietic stem cells according to claim 7, wherein: after the step S6, the method further comprises the step of recovering the cord blood stem cells in the frozen storage bag, specifically, the frozen storage bag is placed in a water bath at 37 ℃ for 2 minutes for recovery.
9. The method for cryopreserving umbilical cord blood hematopoietic stem cells according to claim 8, wherein: after the step of putting the frozen bag in a water bath at 37 ℃ for 2 minutes for resuscitation, the method also comprises the step of determining the viability of the cord blood stem cells.
10. The method for cryopreserving umbilical cord blood hematopoietic stem cells according to claim 9, wherein: in step S6, the frozen storage bag is stored in the environment of the fourth temperature, and the storage time is T0 ≥ 1 month.
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| CN112568219A (en) * | 2020-12-29 | 2021-03-30 | 中博瑞康（北京）生物技术有限公司 | Low-temperature preservation solution for stem cells and umbilical cord blood and preparation method thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1407088A (en) * | 2001-09-06 | 2003-04-02 | 周胜利 | Method for establishing hematopoietic stem cells bank by extracting hematopoietic cells from placenta tissues |
| CN101990888A (en) * | 2009-08-31 | 2011-03-30 | 深圳市北科生物科技有限公司 | Stem cell freezing medium and freezing method of stem cells |
| KR20140059988A (en) * | 2012-11-09 | 2014-05-19 | 주식회사 휴림바이오셀 | Cryopreservation medium for mesenchymal stem cells and cryopreservation method for mesenchymal stem cells using the same |
| CN106982821A (en) * | 2017-05-22 | 2017-07-28 | 安徽瑞杰赛尔生物科技有限公司 | Umbilical cord mesenchymal stem cells clinic freezes protection liquid composition and application thereof |
| CN107711823A (en) * | 2017-11-23 | 2018-02-23 | 深圳市北科生物科技有限公司 | The cells frozen storing liquid and its application that a kind of normal temperature preserves |
| CN108251361A (en) * | 2018-04-19 | 2018-07-06 | 重庆斯德姆生物技术有限公司 | A kind of method that umbilical cord tissue block froze, and recovered and prepared mescenchymal stem cell |
| CN207911916U (en) * | 2017-12-28 | 2018-09-28 | 四川常青树生物科技有限公司 | A kind of stem cell freezen protective case |
| CN109497039A (en) * | 2018-10-29 | 2019-03-22 | 上海慧存医疗科技有限公司 | The cryopreservation resuscitation method of umbilical cord tissue and the preparation method of mescenchymal stem cell |
| CN110839616A (en) * | 2019-11-29 | 2020-02-28 | 安徽惠恩生物科技股份有限公司 | Stem cell refrigerating fluid and refrigerating method thereof |
-
2020
- 2020-06-18 CN CN202010558339.9A patent/CN111685103A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1407088A (en) * | 2001-09-06 | 2003-04-02 | 周胜利 | Method for establishing hematopoietic stem cells bank by extracting hematopoietic cells from placenta tissues |
| CN101990888A (en) * | 2009-08-31 | 2011-03-30 | 深圳市北科生物科技有限公司 | Stem cell freezing medium and freezing method of stem cells |
| KR20140059988A (en) * | 2012-11-09 | 2014-05-19 | 주식회사 휴림바이오셀 | Cryopreservation medium for mesenchymal stem cells and cryopreservation method for mesenchymal stem cells using the same |
| CN106982821A (en) * | 2017-05-22 | 2017-07-28 | 安徽瑞杰赛尔生物科技有限公司 | Umbilical cord mesenchymal stem cells clinic freezes protection liquid composition and application thereof |
| CN107711823A (en) * | 2017-11-23 | 2018-02-23 | 深圳市北科生物科技有限公司 | The cells frozen storing liquid and its application that a kind of normal temperature preserves |
| CN207911916U (en) * | 2017-12-28 | 2018-09-28 | 四川常青树生物科技有限公司 | A kind of stem cell freezen protective case |
| CN108251361A (en) * | 2018-04-19 | 2018-07-06 | 重庆斯德姆生物技术有限公司 | A kind of method that umbilical cord tissue block froze, and recovered and prepared mescenchymal stem cell |
| CN109497039A (en) * | 2018-10-29 | 2019-03-22 | 上海慧存医疗科技有限公司 | The cryopreservation resuscitation method of umbilical cord tissue and the preparation method of mescenchymal stem cell |
| CN110839616A (en) * | 2019-11-29 | 2020-02-28 | 安徽惠恩生物科技股份有限公司 | Stem cell refrigerating fluid and refrigerating method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 钟卫一等: "-80℃冰箱降温-液氮冻存脐血造血干细胞", 《右江民族医学院学报》 * |
| 陈林等: "脐带血造血干细胞分离冻存方法优化", 《重庆理工大学学报(自然科学)》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112568219A (en) * | 2020-12-29 | 2021-03-30 | 中博瑞康（北京）生物技术有限公司 | Low-temperature preservation solution for stem cells and umbilical cord blood and preparation method thereof |
| CN112568219B (en) * | 2020-12-29 | 2024-04-05 | 中博瑞康(上海)生物技术有限公司 | Low-temperature preservation solution for stem cells and umbilical cord blood and preparation method thereof |
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