CN112391342A - Efficient recovery method for umbilical cord blood stem cells - Google Patents
Efficient recovery method for umbilical cord blood stem cells Download PDFInfo
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- CN112391342A CN112391342A CN202011415728.2A CN202011415728A CN112391342A CN 112391342 A CN112391342 A CN 112391342A CN 202011415728 A CN202011415728 A CN 202011415728A CN 112391342 A CN112391342 A CN 112391342A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/42—Organic phosphate, e.g. beta glycerophosphate
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
Abstract
The invention discloses a method for efficiently recovering umbilical cord blood stem cells, which comprises the following operation steps: (1) precooling the cord blood resuscitation protection liquid, then taking the cryopreserved cord blood out of a liquid nitrogen tank, and putting the cord blood into a water bath kettle to slowly shake the water bath for resuscitation; (2) adding the thawed resuscitation cord blood into a resuscitation protection solution, and centrifuging; (3) centrifuging, removing red blood cells and the frozen stock solution, adding resuscitation protection solution again, and centrifuging again; (4) centrifuging, sucking and removing supernatant, removing red blood cells, adding resuscitation protection solution for resuspension, sampling, recording cell viability and counting; (5) continuously adding a resuscitation protection solution into the mixture for centrifugation, discarding supernatant, and adding the resuscitated cord blood stem cells into a culture medium for culture. The method for efficiently recovering the cord blood stem cells can ensure that the cord blood stem cells have higher survival rate in the recovery process, and effectively improves the activity of the cord blood stem cells after recovery.
Description
Technical Field
The invention belongs to the technical field of clinical treatment of cord blood stem cells, and particularly relates to a high-efficiency recovery method of cord blood stem cells.
Background
Stem Cells (SC) are a type of self-replicating pluripotent cells that under certain conditions can differentiate into a variety of functional cells, known in the medical community as "universal cells". Cord blood is blood that remains in the placenta and umbilical cord after the fetus is delivered, ligated and severed, and is typically discarded. The blood in each infant's umbilical cord contains a large number of stem cells, which are one of the major sources of adult stem cells. In the last 70 th century, the umbilical cord blood is found to be rich in stem cells, particularly, the content of hematopoietic stem cells can be comparable to that of bone marrow, and the umbilical cord blood can replace the bone marrow to carry out stem cell transplantation and treat diseases such as leukemia, aplastic anemia and the like. Compared with bone marrow stem cells and peripheral blood stem cells, the neonatal umbilical cord blood stem cells have small foreign body rejection response, low immunogenicity and 10-20 times of the regenerative capacity and speed of the neonatal umbilical cord blood stem cells. In addition, umbilical cord blood also contains Mesenchymal Stem Cells (MSC), and can be differentiated into skin, muscle, bone marrow, cardiac muscle, nerve cells and the like, so that umbilical cord blood becomes an important source of hematopoietic stem cells, is a very important human biological resource, and has remarkable significance and effect.
The cord blood is rich in a large amount of hematopoietic stem cells and can replace bone marrow for stem cell transplantation. At present, more than 80 diseases can be treated medically, the existing cord blood resuscitation protective solution only maintains the osmotic pressure of cells and cannot well maintain the cell state, and the difference of the number of the cells is larger after resuscitation compared with before cryopreservation, so that the cord blood resuscitation protective solution which is efficient, does not contain any animal-derived component, can protect the cells and reduce the cell fragmentation is researched, and the cord blood resuscitation protective solution has good application prospect and economic benefit.
Disclosure of Invention
The invention aims to provide a high-efficiency cord blood stem cell recovery method which has a good recovery effect, is high in efficiency and does not contain any animal-derived components.
The invention is realized by the following technical scheme.
A method for efficiently resuscitating cord blood stem cells comprises the following operation steps:
(1) in order to reduce the temperature difference with cord blood in a melting state, pre-cooling the cord blood resuscitation protection liquid, taking out the cryopreserved cord blood from a liquid nitrogen tank, preheating a water bath to 35-40 ℃, putting the cord blood into the water bath to slowly shake the water bath for resuscitation, wherein the resuscitation protection liquid is prepared from the following components in parts by weight: 40-60 parts of human serum albumin, 410-420 parts of dextran, 20-28 parts of fructose diphosphate sodium and 75-85 parts of PBS buffer solution;
(2) adding the thawed resuscitation cord blood into a resuscitation protection solution, and centrifuging;
(3) centrifuging, removing red blood cells and the frozen stock solution, adding resuscitation protection solution again, and centrifuging again;
(4) centrifuging, sucking and removing supernatant, removing red blood cells, adding resuscitation protection solution for resuspension, sampling, recording cell viability and counting;
(5) continuously adding the resuscitation protection solution into the mixture for centrifugation, discarding the supernatant, repeating the steps for 3-5 times, and adding the resuscitated cord blood stem cells into a culture medium for culture.
Specifically, in the step (1), the temperature of the pre-cooling treatment is 3-5 ℃, and the time of the pre-cooling treatment is 25-35 min.
Specifically, in the step (2), the volume of the resuscitation protection solution is 1.5-2.5 times of the volume of the resuscitation umbilical cord blood.
Specifically, in the steps (2) to (5), the rotation speed of the centrifuge during centrifugation is 1400-1600r/min, the centrifugation time is 8-12min, and the temperature during centrifugation is 3-5 ℃.
Specifically, in the step (4), the sampling counting specifically includes: 1ml of cell suspension was taken out to an EP tube for trypan blue staining counting, and the cell viability was recorded and counted.
According to the technical scheme, the beneficial effects of the invention are as follows:
the method for efficiently recovering the cord blood stem cells is simple and efficient to operate, can ensure that the cord blood stem cells have higher survival rate in the recovery process, and effectively improves the activity of the cord blood stem cells after recovery. The addition of dextran and fructose diphosphate sodium can effectively improve the activity of cells and effectively avoid the loss of the cell activity caused by the change of temperature difference when the temperature of the cells changes; in the invention, the resuscitation protection solution is added to protect the cord blood stem cells in the centrifugation process, so that physical damage to the cord blood stem cells in the centrifugation process can be effectively avoided, and further the cell activity is reduced.
Drawings
FIG. 1 shows the results of apoptosis detection using a flow cytometer on cord blood stem cells recovered in example 1.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
A method for efficiently resuscitating cord blood stem cells comprises the following operation steps:
(1) in order to reduce the temperature difference with cord blood in a melting state, precooling the cord blood resuscitation protection solution at the temperature of 3 ℃ for 25min, then taking out the cryopreserved cord blood from a liquid nitrogen tank, preheating a water bath to 35 ℃, putting the cord blood into the water bath to resuscitate by slowly shaking a water bath, wherein the resuscitation protection solution is prepared from the following components in parts by weight: 40 parts of human serum albumin, 410 parts of dextran, 20 parts of fructose diphosphate sodium and 75 parts of PBS buffer solution;
(2) adding the thawed resuscitated umbilical cord blood into a resuscitating protective solution with the volume 1.5 times of the volume of the resuscitated umbilical cord blood, and centrifuging at the rotating speed of 1400r/min for 8min at the centrifugation temperature of 3 ℃;
(3) after centrifugation, sucking and removing supernatant, removing red blood cells and frozen stock solution, adding resuscitation protection solution again, and centrifuging again, wherein the rotating speed of the centrifuge is 1400r/min, the centrifuging time is 8min, and the temperature during centrifugation is 3 ℃;
(4) centrifuging, sucking and removing supernatant, removing red blood cells, adding resuscitation protection solution for resuspension, taking out 1ml of cell suspension, placing the cell suspension into an EP tube for trypan blue staining counting, recording the cell viability and counting according to the counting result;
(5) continuously adding the resuscitation protection solution into the mixture for centrifugation, discarding the supernatant, wherein the rotation speed of a centrifuge is 1400r/min during centrifugation, the centrifugation time is 8min, the temperature during centrifugation is 3 ℃, repeating the steps for 3 times, and then adding the resuscitated umbilical cord blood stem cells into a culture medium for culture.
Example 2
A method for efficiently resuscitating cord blood stem cells comprises the following operation steps:
(1) in order to reduce the temperature difference with cord blood in a melting state, precooling the cord blood resuscitation protection solution at the temperature of 4 ℃ for 30min, then taking out the cryopreserved cord blood from a liquid nitrogen tank, preheating a water bath to 38 ℃, putting the cord blood into the water bath to resuscitate by slowly shaking a water bath, wherein the resuscitation protection solution is prepared from the following components in parts by weight: 50 parts of human serum albumin, 415 parts of dextran, 24 parts of fructose diphosphate sodium and 80 parts of PBS buffer solution;
(2) adding the thawed resuscitated umbilical cord blood into a resuscitating protective solution with the volume 2.0 times of that of the umbilical cord blood, and centrifuging at the rotating speed of 1500r/min for 10min at the temperature of 4 ℃;
(3) after centrifugation, sucking and removing supernatant, removing red blood cells and frozen stock solution, adding resuscitation protection solution again, and centrifuging again, wherein the rotating speed of a centrifuge is 1500r/min, the centrifuging time is 10min, and the temperature during centrifugation is 4 ℃;
(4) centrifuging, sucking and removing supernatant, removing red blood cells, adding resuscitation protection solution for resuspension, taking out 1ml of cell suspension, placing the cell suspension into an EP tube for trypan blue staining counting, recording the cell viability and counting according to the counting result;
(5) continuously adding the resuscitation protection solution into the mixture for centrifugation, discarding the supernatant, repeating the steps for 4 times at the rotation speed of 1500r/min and the centrifugation time of 10min and the centrifugation temperature of 4 ℃, and adding the resuscitated cord blood stem cells into the culture medium for culture.
Example 3
A method for efficiently resuscitating cord blood stem cells comprises the following operation steps:
(1) in order to reduce the temperature difference with cord blood in a melting state, precooling the cord blood resuscitation protection solution at the temperature of 5 ℃ for 35min, then taking out the cryopreserved cord blood from a liquid nitrogen tank, preheating a water bath to 40 ℃, putting the cord blood into the water bath to resuscitate by slowly shaking a water bath, wherein the resuscitation protection solution is prepared from the following components in parts by weight: 60 parts of human serum albumin, 420 parts of dextran, 28 parts of fructose diphosphate sodium and 85 parts of PBS buffer solution;
(2) adding the thawed resuscitated umbilical cord blood into a resuscitating protective solution with the volume 2.5 times of that of the umbilical cord blood, and centrifuging at the rotating speed of 1600r/min for 12min at the temperature of 5 ℃;
(3) after centrifugation, sucking and removing supernatant, removing red blood cells and frozen stock solution, adding resuscitation protection solution again, and centrifuging again, wherein the rotating speed of a centrifuge is 1600r/min, the centrifuging time is 12min, and the temperature during centrifugation is 5 ℃;
(4) centrifuging, sucking and removing supernatant, removing red blood cells, adding resuscitation protection solution for resuspension, taking out 1ml of cell suspension, placing the cell suspension into an EP tube for trypan blue staining counting, recording the cell viability and counting according to the counting result;
(5) continuously adding the resuscitation protection solution into the mixture for centrifugation, discarding the supernatant, wherein the rotation speed of a centrifuge is 1600r/min, the centrifugation time is 12min, the temperature during centrifugation is 5 ℃, repeating the steps for 5 times, and then adding the resuscitated umbilical cord blood stem cells into a culture medium for culture.
Comparative example 1
In the step (1), the resuscitation protection solution does not contain fructose diphosphate sodium, and the rest of the operation steps are completely the same as those in the example 1.
Comparative example 2
During centrifugation in the step (2), the resuscitation protection solution is replaced by the same amount of physiological saline, and the rest of the operation steps are completely the same as those in the example 2.
The cord blood stem cells are efficiently recovered by the methods of each example and each comparative example, and then the cell viability rate after recovery is detected, and the test results are shown in table 1:
TABLE 1 survival Rate of cord blood Stem cells after Resuscitation
| Item | Cell viability% |
| Example 1 | 93 |
| Comparative example 1 | 81 |
| Example 2 | 94 |
| Comparative example 2 | 85 |
| Example 3 | 96 |
As can be seen from Table 1, the method for efficiently recovering cord blood stem cells provided by the invention can effectively improve the survival rate of cord blood stem cells after recovery.
The cord blood stem cells recovered in example 1 were subjected to apoptosis detection using a flow cytometer, and the detection results are shown in fig. 1, where the recovered mesenchymal stem cells have fewer dead cells, fewer apoptotic aged cells, and a good cell state.
The number of lymphocytes is detected by a hemocytometer, the number of lymphocytes before recovery in example 1 is compared with the number of lymphocytes after recovery, and the recovery ratio is calculated, as shown in table 2, it is found that the recovery rate of the lymphocytes in the example is high, the cell damage is small and can reach more than 70%, while the recovery rate of the lymphocytes in the comparative example is only about 54%, and the method in example 1 is further verified to have a protective effect on cell recovery.
TABLE 2 high lymphocyte recovery
It is to be understood that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and those skilled in the art should understand that they can make various changes, modifications, additions and substitutions within the spirit and scope of the present invention.
Claims (5)
1. A method for efficiently resuscitating cord blood stem cells is characterized by comprising the following operation steps:
(1) precooling the cord blood resuscitation protection liquid, then taking out the cryopreserved cord blood from a liquid nitrogen tank, preheating a water bath to 35-40 ℃, putting the cord blood into the water bath to slowly shake the water bath for resuscitation, wherein the resuscitation protection liquid is prepared from the following components in parts by weight: 40-60 parts of human serum albumin, 410-420 parts of dextran, 20-28 parts of fructose diphosphate sodium and 75-85 parts of PBS buffer solution;
(2) adding the thawed resuscitation cord blood into a resuscitation protection solution, and centrifuging;
(3) centrifuging, removing red blood cells and the frozen stock solution, adding resuscitation protection solution again, and centrifuging again;
(4) centrifuging, sucking and removing supernatant, removing red blood cells, adding resuscitation protection solution for resuspension, sampling, recording cell viability and counting;
(5) continuously adding the resuscitation protection solution into the mixture for centrifugation, discarding the supernatant, repeating the steps for 3-5 times, and adding the resuscitated cord blood stem cells into a culture medium for culture.
2. The method for resuscitating umbilical cord blood stem cells with high efficiency as claimed in claim 1, wherein in step (1), the temperature of the pre-cooling treatment is 3-5 ℃ and the time of the pre-cooling treatment is 25-35 min.
3. The method for efficiently resuscitating cord blood stem cells according to claim 1, wherein in step (2), the volume of the resuscitation protection solution is 1.5-2.5 times the volume of the resuscitated cord blood.
4. The method for resuscitating cord blood stem cells of claim 1, wherein the rotation speed of the centrifuge is 1400-1600r/min, the centrifugation time is 8-12min, and the centrifugation temperature is 3-5 ℃ in the steps (2) - (5).
5. The method for resuscitating umbilical cord blood stem cells with high efficiency as claimed in claim 1, wherein the sampling and counting in step (4) is performed by: 1ml of cell suspension was taken out to an EP tube for trypan blue staining counting, and the cell viability was recorded and counted.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113293136A (en) * | 2021-05-24 | 2021-08-24 | 成都新生命霍普医学检验实验室有限公司 | Method for reviving cells and application |
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